TWI723065B - Medical use of hydroxypurine compound - Google Patents

Medical use of hydroxypurine compound Download PDF

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TWI723065B
TWI723065B TW105135042A TW105135042A TWI723065B TW I723065 B TWI723065 B TW I723065B TW 105135042 A TW105135042 A TW 105135042A TW 105135042 A TW105135042 A TW 105135042A TW I723065 B TWI723065 B TW I723065B
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methyl
purine
dimethyl
dione
ethyl
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TW201717956A (en
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吳凌云
張鵬
張麗
誌中 陳
曙輝 陳
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大陸商廣東衆生睿創生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/04—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms

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Abstract

The invention discloses applications of a series of hydroxypurine compounds in preparation of drugs for treating or preventing liver diseases, and specifically discloses uses of a compound having a structure as shown in Formula (I), tautomers and pharmaceutically acceptable salts thereof in drugs of liver diseases.

Description

羥基嘌呤類化合物的醫藥用途Medical use of hydroxypurine compounds

本發明涉及一系列羥基嘌呤類化合物在製備治療肝病藥物中的應用，具體涉及式(I)所示化合物、其互變異構體或其藥學上可接受的鹽在製備治療肝病藥物中的應用。 The present invention relates to the application of a series of hydroxypurine compounds in the preparation of drugs for treating liver diseases, in particular to the use of compounds represented by formula (I), their tautomers or their pharmaceutically acceptable salts in the preparation of drugs for treating liver diseases.

磷酸二酯酶(PDE)催化水解環化核苷酸cGMP和cAMP，通過控制這兩個重要的二級信號因子的分子內濃度調控各種生理反應。環化核苷酸cGMP和cAMP分子內的調控異常是導致許多疾病的原因，現在已經有多個藥物通過抑制PDE活性來改善和治療疾病，如PDE5抑制劑用於肺動脈高壓，PDE4抑制劑用於銀屑病引起的關節炎。目前已知的磷酸二酯酶基因共有十一個大類，每一類又可以表達若干亞型，總共有超過100種PDE亞型。不同的亞型具有不同的結構，不同的組織分布，對環化核苷酸cGMP和cAMP的活性也有很大的不同，調控的生理功能也是千差萬別。 Phosphodiesterase (PDE) catalyzes the hydrolysis of cyclized nucleotides cGMP and cAMP, and regulates various physiological reactions by controlling the intramolecular concentration of these two important secondary signal factors. Abnormal regulation of the cyclic nucleotide cGMP and cAMP molecules is the cause of many diseases. There are now many drugs that inhibit PDE activity to improve and treat diseases, such as PDE5 inhibitors for pulmonary hypertension and PDE4 inhibitors for pulmonary hypertension. Arthritis caused by psoriasis. There are currently eleven major classes of phosphodiesterase genes, each of which can express several subtypes. There are more than 100 PDE subtypes in total. Different subtypes have different structures, different tissue distributions, and the activities of cyclic nucleotide cGMP and cAMP are also very different, and the physiological functions of regulation are also very different.

PDE2磷酸二酯酶可以催化水解環化核苷酸cGMP和cAMP，同時cAMP活性受cGMP調控，對於細胞內的cGMP和cAMP功能平衡起關鍵作用。PDE2在人體組織中廣泛表達，分布主要是心臟、中樞神經系統、肝臟、腎上腺、內皮細胞、和血小板等。PDE2參與調節各項生理活性，如中樞的學習、記憶和認知等過程，維持心臟、平滑肌和內皮細胞的基本節律、內 皮細胞的通透性、調節炎症反應。PDE2基因敲初小鼠直接導致胚胎死亡。通過抑制PDE2活性可能用於各種中樞，心血管疾病，和控制炎症反應。 PDE2 phosphodiesterase can catalyze the hydrolysis of cyclized nucleotides cGMP and cAMP. At the same time, cAMP activity is regulated by cGMP, which plays a key role in the functional balance of cGMP and cAMP in cells. PDE2 is widely expressed in human tissues, mainly distributed in the heart, central nervous system, liver, adrenal glands, endothelial cells, and platelets. PDE2 participates in the regulation of various physiological activities, such as central learning, memory and cognition processes, and maintains the basic rhythms and internal rhythms of the heart, smooth muscle and endothelial cells. Permeability of skin cells, regulate inflammation. PDE2 knockout mice directly lead to embryonic death. By inhibiting the activity of PDE2, it may be used for various central, cardiovascular diseases, and control inflammation.

多種天然和合成的嘌呤類化合物的非選擇性PDE抑制活性很早就被發現，如咖啡因，茶鹼，己酮可可鹼等。己酮可可鹼(PDE2活性)臨床上批准用於周邊血管諸塞造成的下肢跛行，主要作用是降低血液黏度，提高紅細胞變形，抑制血小板凝聚等。新型的高選擇性PDE2抑制劑也有報導用於控制內皮細胞的分裂和血管再生，和改善中樞認知障礙。但總體新型的選擇性PDE2抑制劑的開發和應用還非常有限，發現和應用新型PDE2抑制劑具有廣闊的前景。 The non-selective PDE inhibitory activity of a variety of natural and synthetic purine compounds has been discovered very early, such as caffeine, theophylline, and pentoxifylline. Pentoxifylline (PDE2 activity) is clinically approved for lower limb claudication caused by peripheral vascular congestion. Its main function is to reduce blood viscosity, increase red blood cell deformation, and inhibit platelet aggregation. New and highly selective PDE2 inhibitors have also been reported to be used to control endothelial cell division and angiogenesis, and to improve central cognitive impairment. However, the overall development and application of novel selective PDE2 inhibitors is still very limited, and the discovery and application of novel PDE2 inhibitors has broad prospects.

腫瘤壞死因子α(tumor necrosis factor alpha，TNF□α)是一種具有多種生物學活性的細胞因子，對多種疾病特別是免疫和炎症相關的疾病的發生、發展及治療具有重要影響。TNF□α主要由單核細胞和巨噬細胞系產生，參與機體的免疫調節和細胞因子網絡協調。正常情况下，TNF□α對免疫防禦和免疫監督起著重要作用，但在某些情况下却有不良作用。研究顯示，TNF□α過量表達可誘導促炎細胞因子如白介素1(interleukon□1，IL□1)、IL□6等的表達、增加內皮細胞通透性、上調黏附分子表達、激活中性白細胞和嗜酸細胞，並且誘導骨滑膜細胞和軟骨細胞分泌急性期物質和組織降解酶等促進炎症的發生。這些病理反應在許多免疫介導的炎症性疾病(Immune□mediated inflammatory diseases，IMID)的發生發展中起著非常重要的作用，如風濕性關節炎(rheumatoid arthritis，RA)、牛皮癬關節炎(psoriatic arthritis，PsA)、僵直性脊椎炎(ankylosing spondylitis，AS)、炎症性腸炎(inflammatory bowel disease，IBD)、幼年型慢性關節炎(juvenile chronic arthritis，JCA)以及血管炎(vasculitis)等。研究表明，TNF□α是以上多種IMID的理想靶標，同時對於一些由於長期慢性炎症損傷造成的疾 病，如脂肪肝炎、慢性阻塞性肺炎等，使用TNF□α抑制藥物(TNF□α inhibitors)來中和過量的TNF□α，也是有效的防治和治療途徑。TNF□α單抗藥物在臨床上已經證明抑制TNF□α是非常有效的治療上述炎症相關疾病的手段。PDE2從機理上可以調控TNF□α的表達，因此可以通過調節PDE2活性了控制TNF□α的水平，從而可以實現控制炎症反應。 Tumor necrosis factor alpha (TNF□α) is a cytokine with a variety of biological activities, which has an important impact on the occurrence, development and treatment of various diseases, especially immune and inflammation-related diseases. TNF□α is mainly produced by monocytes and macrophage cell lines, and participates in the body's immune regulation and cytokine network coordination. Under normal circumstances, TNF□α plays an important role in immune defense and immune supervision, but in some cases it has an adverse effect. Studies have shown that overexpression of TNF□α can induce the expression of pro-inflammatory cytokines such as interleukon□1 (IL□1), IL□6, etc., increase the permeability of endothelial cells, up-regulate the expression of adhesion molecules, and activate neutrophils And eosinophils, and induce bone synovial cells and chondrocytes to secrete acute phase substances and tissue degrading enzymes to promote inflammation. These pathological reactions play a very important role in the occurrence and development of many immune-mediated inflammatory diseases (Immune□mediated inflammatory diseases, IMID), such as rheumatoid arthritis (RA) and psoriatic arthritis , PsA), ankylosing spondylitis (AS), inflammatory bowel disease (IBD), juvenile chronic arthritis (JCA) and vasculitis (vasculitis). Studies have shown that TNF□α is an ideal target for the above multiple IMIDs, and at the same time for some diseases caused by long-term chronic inflammation. For diseases such as steatohepatitis and chronic obstructive pneumonia, the use of TNF□α inhibitors to neutralize excess TNF□α is also an effective way of prevention and treatment. TNF□α monoclonal antibody drugs have been clinically proven to inhibit TNF□α as a very effective treatment for the above-mentioned inflammation-related diseases. PDE2 can regulate the expression of TNF□α in mechanism, so the level of TNF□α can be controlled by regulating the activity of PDE2, so as to control the inflammatory response.

本發明提供式(I)所示化合物、其互變異構體或其藥學上可接受的鹽在製備治療或預防肝病藥物中的應用，

Figure 105135042-A0305-02-0005-4
其中，可將結構單元
Figure 105135042-A0305-02-0005-5
替換為
Figure 105135042-A0305-02-0005-7
，具體替換為
Figure 105135042-A0305-02-0005-8
；L11選自空、C(R)(R’)；R、R’分別獨立地選自H、鹵素、 OH、NH2、CN、任選被取代的1~6元烷基或雜烷基；任選地，R、R’可以環化成3-6元環烷基、雜環烷基；A為空，或選自任選被取代的環烷基、雜環烷基、芳基、雜芳基；L12選自任選被取代的1~6元烷基或雜烷基；R1選自任選被取代的1~6元烷基、3-6元環烷基或雜烷基；“雜”代表N、O、S、C(=O)、S(=O)、S(=O)2，每個基團上雜原子的數目選自1、2、3或4。 The present invention provides the use of a compound represented by formula (I), its tautomer or a pharmaceutically acceptable salt thereof in the preparation of a medicine for treating or preventing liver disease,
Figure 105135042-A0305-02-0005-4
 Among them, the structural unit can be
Figure 105135042-A0305-02-0005-5
Replace with
Figure 105135042-A0305-02-0005-7
, Specifically replaced with
Figure 105135042-A0305-02-0005-8
; L 11 is selected from empty, C(R)(R'); R and R'are independently selected from H, halogen, OH, NH 2 , CN, optionally substituted 1~6 membered alkyl or heteroalkane Optionally, R, R'can be cyclized into 3-6 membered cycloalkyl, heterocycloalkyl; A is empty, or selected from optionally substituted cycloalkyl, heterocycloalkyl, aryl, Heteroaryl; L 12 is selected from optionally substituted 1 to 6 membered alkyl or heteroalkyl; R 1 is selected from optionally substituted 1 to 6 membered alkyl, 3-6 membered cycloalkyl or heteroalkyl Group; "Hetero" represents N, O, S, C(=O), S(=O), S(=O) 2 , and the number of heteroatoms on each group is selected from 1, 2, 3, or 4.

本發明的一些方案中，上述肝病選自非酒精性脂肪肝和肝纖維化。 In some aspects of the present invention, the aforementioned liver disease is selected from non-alcoholic fatty liver and liver fibrosis.

本發明的一些方案中，上述R、R’、A、L12、R1中取代基分別獨立地選自鹵素、OH、NH2、CN、任選被取代的1~6元烷基、3-6元環烷基或雜烷基，每個基團取代基的數目分別獨立地選自1、2或3。 In some embodiments of the present invention, the substituents in R, R', A, L 12 and R 1 are independently selected from halogen, OH, NH 2 , CN, optionally substituted 1- to 6-membered alkyl, 3 6-membered cycloalkyl or heteroalkyl, the number of substituents in each group is independently selected from 1, 2, or 3.

本發明的一些方案中，上述R、R’、A、L12、R1中取代基分別獨立 地選自鹵素、CF3、CN、OH、Me、Et、正丙基、異丙基、環丙基、

Figure 105135042-A0305-02-0006-15
、
Figure 105135042-A0305-02-0006-16
。 In some embodiments of the present invention, the substituents in R, R', A, L 12 and R 1 are independently selected from halogen, CF 3 , CN, OH, Me, Et, n-propyl, isopropyl, ring Propyl,
Figure 105135042-A0305-02-0006-15
,
Figure 105135042-A0305-02-0006-16
.

本發明的一些方案中，上述R、R’分別獨立地選自H、Me、CF3、Et。 In some aspects of the present invention, the above-mentioned R and R'are independently selected from H, Me, CF 3 , and Et.

本發明的一些方案中，上述L11選自

Figure 105135042-A0305-02-0006-10
、
Figure 105135042-A0305-02-0006-11
、
Figure 105135042-A0305-02-0006-13
、
Figure 105135042-A0305-02-0006-14
、
Figure 105135042-A0305-02-0006-9
 In some aspects of the present invention, the above-mentioned L 11 is selected from
Figure 105135042-A0305-02-0006-10
,
Figure 105135042-A0305-02-0006-11
,
Figure 105135042-A0305-02-0006-13
,
Figure 105135042-A0305-02-0006-14
,
Figure 105135042-A0305-02-0006-9

本發明的一些方案中，上述A選自任選被取代的：3~12元環烷基或雜環烷基、5~12元芳基或雜芳基。 In some aspects of the present invention, the above A is selected from optionally substituted: 3-12 membered cycloalkyl or heterocycloalkyl, 5-12 membered aryl or heteroaryl.

本發明的一些方案中，上述A選自任選被取代的：環丙基、環丁基、環戊基、環己基、環氧戊基、苯基、吡啶基、吡嗪基、噁唑基、異噁唑基、噻唑基、雙環[1.1.1]戊烷，或選自由上述基團中任意兩個組成的二聯環基、螺環基或並環基。 In some aspects of the present invention, the above A is selected from optionally substituted: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, epoxypentyl, phenyl, pyridyl, pyrazinyl, oxazolyl , Isoxazolyl, thiazolyl, bicyclo[1.1.1]pentane, or a bicyclic group, spirocyclic group or pentacyclic group consisting of any two of the above groups.

本發明的一些方案中，上述A選自任選被取代的：

Figure 105135042-A0305-02-0006-22
、
Figure 105135042-A0305-02-0006-21
、
Figure 105135042-A0305-02-0006-18
 In some aspects of the present invention, the above-mentioned A is selected from optionally substituted:
Figure 105135042-A0305-02-0006-22
,
Figure 105135042-A0305-02-0006-21
,
Figure 105135042-A0305-02-0006-18

本發明的一些方案中，上述A選自

Figure 105135042-A0305-02-0006-27
、
Figure 105135042-A0305-02-0006-26
、
Figure 105135042-A0305-02-0006-24
、
Figure 105135042-A0305-02-0006-20
Figure 105135042-A0305-02-0007-31
 In some aspects of the present invention, the above A is selected from
Figure 105135042-A0305-02-0006-27
,
Figure 105135042-A0305-02-0006-26
,
Figure 105135042-A0305-02-0006-24
,
Figure 105135042-A0305-02-0006-20
Figure 105135042-A0305-02-0007-31

本發明的一些方案中，上述L12選自亞甲基、

Figure 105135042-A0305-02-0007-38
、
Figure 105135042-A0305-02-0007-37
、
Figure 105135042-A0305-02-0007-28
 In some aspects of the present invention, the above-mentioned L 12 is selected from methylene,
Figure 105135042-A0305-02-0007-38
,
Figure 105135042-A0305-02-0007-37
,
Figure 105135042-A0305-02-0007-28

本發明的一些方案中，上述R1選自Me、CHF2、CF3、Et、CH2CF3、 異丙基、

Figure 105135042-A0305-02-0007-32
、環丙基、
Figure 105135042-A0305-02-0007-33
、
Figure 105135042-A0305-02-0007-34
、
Figure 105135042-A0305-02-0007-35
、
Figure 105135042-A0305-02-0007-36
。 In some aspects of the present invention, the above-mentioned R 1 is selected from Me, CHF 2 , CF 3 , Et, CH 2 CF 3 , isopropyl,
Figure 105135042-A0305-02-0007-32
, Cyclopropyl,
Figure 105135042-A0305-02-0007-33
,
Figure 105135042-A0305-02-0007-34
,
Figure 105135042-A0305-02-0007-35
,
Figure 105135042-A0305-02-0007-36
.

本發明選自下式化合物在製備治療或預防肝病藥物中的應用：

Figure 105135042-A0305-02-0007-29
Figure 105135042-A0305-02-0008-39
 The present invention is selected from the application of compounds of the following formulae in the preparation of drugs for treating or preventing liver diseases:
Figure 105135042-A0305-02-0007-29
Figure 105135042-A0305-02-0008-39

進一步，本發明選自下式化合物在製備治療或預防肝病藥物中應 用：

Figure 105135042-A0305-02-0009-239
Figure 105135042-A0305-02-0009-238
 Further, the compounds of the present invention are selected from the following formulae for use in the preparation of drugs for treating or preventing liver diseases:
Figure 105135042-A0305-02-0009-239
Figure 105135042-A0305-02-0009-238

相關定義。 Related definitions.

除非另有說明，本文所用的下列術語和短語旨在具有下列含義。一個特定的術語或短語在沒有特別定義的情况下不應該被認為是不確定的或不清楚的，而應該按照普通的含義去理解。當本文中出現商品名時，意在指代其對應的商品或其活性成分。 Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A specific term or phrase should not be considered uncertain or unclear without a special definition, but should be understood in its ordinary meaning. When a trade name appears in this article, it is meant to refer to its corresponding commodity or its active ingredient.

這裡所採用的術語“藥學上可接受的”，是針對那些化合物、材料、組合物和/或劑型而言，它們在可靠的醫學判斷的範圍之內，適用於與人類 和動物的組織接觸使用，而沒有過多的毒性、刺激性、過敏性反應或其它問題或並發症，與合理的利益/風險比相稱。 The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and/or dosage forms that are within the scope of reliable medical judgment and are suitable for use with humans. Use in contact with animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.

術語“藥學上可接受的鹽”是指本發明化合物的鹽，由本發明發現的具有特定取代基的化合物與相對無毒的酸或鹼製備。當本發明的化合物中含有相對酸性的功能團時，可以通過在純的溶液或合適的惰性溶劑中用足夠量的鹼與這類化合物的中性形式接觸的方式獲得鹼加成鹽。藥學上可接受的鹼加成鹽包括鈉、鉀、鈣、銨、有機氨或鎂鹽或類似的鹽。當本發明的化合物中含有相對鹼性的官能團時，可以通過在純的溶液或合適的惰性溶劑中用足夠量的酸與這類化合物的中性形式接觸的方式獲得酸加成鹽。藥學上可接受的酸加成鹽的實例包括無機酸鹽，所述無機酸包括例如鹽酸、氫溴酸、硝酸、碳酸，碳酸氫根，磷酸、磷酸一氫根、磷酸二氫根、硫酸、硫酸氫根、氫碘酸、亞磷酸等；以及有機酸鹽，所述有機酸包括如乙酸、丙酸、異丁酸、馬來酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、鄰苯二甲酸、苯磺酸、對甲苯磺酸、檸檬酸、酒石酸和甲磺酸等類似的酸；還包括氨基酸(如精氨酸等)的鹽，以及如葡糖醛酸等有機酸的鹽(參見Berge et al.,"Pharmaceutical Salts",Journal of Pharmaceutical Science 66：1-19(1977))。本發明的某些特定的化合物含有鹼性和酸性的官能團，從而可以被轉換成任一鹼或酸加成鹽。 The term "pharmaceutically acceptable salt" refers to a salt of the compound of the present invention, which is prepared from the compound with specific substituents discovered in the present invention and a relatively non-toxic acid or base. When the compound of the present invention contains a relatively acidic functional group, the base addition salt can be obtained by contacting the neutral form of the compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts. When the compound of the present invention contains a relatively basic functional group, the acid addition salt can be obtained by contacting the neutral form of the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, the organic acid includes, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; also include salts of amino acids (such as arginine, etc.) , And salts of organic acids such as glucuronic acid (see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specific compounds of the present invention contain basic and acidic functional groups, which can be converted into any base or acid addition salt.

優選地，以常規方式使鹽與鹼或酸接觸，再分離母體化合物，由此再生化合物的中性形式。化合物的母體形式與其各種鹽的形式的不同之處在於某些物理性質，例如在極性溶劑中的溶解度不同。 Preferably, the salt is contacted with a base or acid in a conventional manner, and the parent compound is separated, thereby regenerating the neutral form of the compound. The parent form of the compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents.

本文所用的“藥學上可接受的鹽”屬本發明化合物的衍生物，其中，通過與酸成鹽或與鹼成鹽的方式修飾所述母體化合物。藥學上可接受的鹽的實例包括但不限於：鹼基比如胺的無機酸或有機酸鹽、酸根比如羧酸的 鹼金屬或有機鹽等等。藥學上可接受的鹽包括常規的無毒性的鹽或母體化合物的四級銨鹽，例如無毒的無機酸或有機酸所形成的鹽。常規的無毒性的鹽包括但不限於那些衍生自無機酸和有機酸的鹽，所述的無機酸或有機酸選自2-乙醯氧基苯甲酸、2-羥基乙磺酸、乙酸、抗壞血酸、苯磺酸、苯甲酸、碳酸氫根、碳酸、檸檬酸、依地酸、乙烷二磺酸、乙烷磺酸、富馬酸、葡庚糖、葡糖酸、谷氨酸、乙醇酸、氫溴酸、鹽酸、氫碘酸鹽、羥基、羥萘、羥乙磺酸、乳酸、乳糖、十二烷基磺酸、馬來酸、蘋果酸、扁桃酸、甲烷磺酸、硝酸、草酸、雙羥萘酸、泛酸、苯乙酸、磷酸、多聚半乳糖醛、丙酸、水楊酸、硬脂酸、亞乙酸、琥珀酸、氨基磺酸、對氨基苯磺酸、硫酸、單寧、酒石酸和對甲苯磺酸。 The "pharmaceutically acceptable salt" used herein is a derivative of the compound of the present invention, wherein the parent compound is modified by forming a salt with an acid or a salt with a base. Examples of pharmaceutically acceptable salts include, but are not limited to: inorganic or organic acid salts of bases such as amines, acid radicals such as carboxylic acids Alkali metal or organic salt, etc. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of parent compounds, such as salts formed by non-toxic inorganic or organic acids. Conventional non-toxic salts include, but are not limited to, those derived from inorganic acids and organic acids. The inorganic or organic acids are selected from 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, and ascorbic acid. , Benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptose, gluconic acid, glutamic acid, glycolic acid , Hydrobromic acid, hydrochloric acid, hydroiodide, hydroxyl, hydroxynaphthalene, isethionic acid, lactic acid, lactose, dodecylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, nitric acid, oxalic acid , Pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, sulfamic acid, p-aminobenzenesulfonic acid, sulfuric acid, tannin , Tartaric acid and p-toluenesulfonic acid.

本發明的藥學上可接受的鹽可由含有酸根或鹼基的母體化合物通過常規化學方法合成。一般情况下，這樣的鹽的製備方法是：在水或有機溶劑或兩者的混合物中，經由游離酸或鹼形式的這些化合物與化學計量的適當的鹼或酸反應來製備。一般地，優選醚、乙酸乙酯、乙醇、異丙醇或乙腈等非水介質。 The pharmaceutically acceptable salt of the present invention can be synthesized from the parent compound containing acid or base by conventional chemical methods. In general, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of appropriate base or acid in water or organic solvent or a mixture of both. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

除了鹽的形式，本發明所提供的化合物還存在前藥形式。本文所描述的化合物的前藥容易地在生理條件下發生化學變化從而轉化成本發明的化合物。此外，前體藥物可以在體內環境中通過化學或生化方法被轉換到本發明的化合物。 In addition to salt forms, the compounds provided by the present invention also exist in prodrug forms. The prodrugs of the compounds described herein easily undergo chemical changes under physiological conditions to transform into the compounds of the invention. In addition, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in the in vivo environment.

本發明的某些化合物可以以非溶劑化形式或者溶劑化形式存在，包括水合物形式。一般而言，溶劑化形式與非溶劑化的形式相當，都包含在本發明的範圍之內。 Certain compounds of the present invention may exist in unsolvated or solvated forms, including hydrated forms. Generally speaking, the solvated form is equivalent to the unsolvated form, and both are included in the scope of the present invention.

本發明的某些化合物可以具有不對稱碳原子(光學中心)或雙鍵。外消旋體、非對映異構體、幾何異構體和單個的異構體都包括在本發明的範圍之內。 Certain compounds of the present invention may have asymmetric carbon atoms (optical centers) or double bonds. Racemates, diastereomers, geometric isomers and individual isomers are all included in the scope of the present invention.

除非另有說明，用楔形鍵和虛綫鍵(

Figure 105135042-A0305-02-0012-241
)表示一個立體中心的絕對構型，用
Figure 105135042-A0305-02-0012-240
表示一個立體中心的相對構型。當本文所述化合物含有烯屬雙鍵或其它幾何不對稱中心，除非另有規定，它們包括E、Z幾何異構體。同樣地，所有的互變異構形式均包括在本發明的範圍之內。 Unless otherwise specified, use wedge keys and dotted keys (
Figure 105135042-A0305-02-0012-241
) Represents the absolute configuration of a three-dimensional center, using
Figure 105135042-A0305-02-0012-240
Represents the relative configuration of a three-dimensional center. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise specified, they include E and Z geometric isomers. Likewise, all tautomeric forms are included within the scope of the present invention.

本發明的化合物可以存在特定的幾何或立體異構體形式。本發明設想所有的這類化合物，包括順式和反式異構體、(-)-和(+)-對映體、(R)-和(S)-對映體、非對映異構體、(D)-異構體、(L)-異構體，及其外消旋混合物和其他混合物，例如對映異構體或非對映體富集的混合物，所有這些混合物都屬於本發明的範圍之內。烷基等取代基中可存在另外的不對稱碳原子。所有這些異構體以及它們的混合物，均包括在本發明的範圍之內。 The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, ( R )- and ( S )-enantiomers, diastereomers Isomers, ( D )-isomers, ( L )-isomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomer-enriched mixtures, all of these mixtures belong to this Within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All these isomers and their mixtures are included in the scope of the present invention.

可以通過的手性合成或手性試劑或者其他常規技術製備光學活性的(R)-和(S)-異構體以及D和L異構體。如果想得到本發明某化合物的一種對映體，可以通過不對稱合成或者具有手性助劑的衍生作用來製備，其中將所得非對映體混合物分離，並且輔助基團裂開以提供純的所需對映異構體。或者，當分子中含有鹼性官能團(如氨基)或酸性官能團(如羧基)時，與適當的光學活性的酸或鹼形成非對映異構體的鹽，然後通過本領域所公知的常用方法進行非對映異構體拆分，然後回收得到純的對映體。此外，對映異構體和非對映異構體的分離通常是通過使用色譜法完成的，所述色譜法採用手性固定相，並任選地與化學衍生法相結合(例如由胺生成氨基甲酸鹽)。 The optically active (R )- and ( S )-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one wants to obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure all Enantiomers are required. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), it forms a diastereomeric salt with an appropriate optically active acid or base, and then passes through a common method known in the art The diastereoisomers are resolved, and then the pure enantiomers are recovered. In addition, the separation of enantiomers and diastereomers is usually accomplished through the use of chromatography, which uses a chiral stationary phase and is optionally combined with chemical derivatization (for example, the formation of amino groups from amines). Formate).

本發明的化合物可以在一個或多個構成該化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素標記化合物，比如氚(3H)，碘-125(125I)或C-14(14C)。本發明的化合物的所有同位素組成的變換，無論放射性與否，都包括在本發明的範圍之內。 The compound of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms constituting the compound. For example, compounds can be labeled with radioisotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C). All changes in the isotopic composition of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.

術語“藥學上可接受的載體”是指能夠遞送本發明有效量活性物質、不干擾活性物質的生物活性並且對宿主或者患者無毒副作用的任何製劑或載體介質代表性的載體包括水、油、蔬菜和礦物質、膏基、洗劑基質、軟膏基質等。這些基質包括懸浮劑、增黏劑、透皮促進劑等。它們的製劑為化妝品領域或局部藥物領域的技術人員所周知。關於載體的其他信息，可以參考Remington：The Science and Practice of Pharmacy,21 st Ed.,Lippincott,Williams & Wilkins(2005)，該文獻的內容通過引用的方式併入本文。 The term "pharmaceutically acceptable carrier" refers to any preparation or carrier medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects to the host or patient. Representative carriers include water, oil, and vegetables. And minerals, cream base, lotion base, ointment base, etc. These bases include suspending agents, tackifiers, penetration enhancers, and the like. Their formulations are well known to those skilled in the field of cosmetics or topical medicine. For other information about the carrier, you can refer to Remington: The Science and Practice of Pharmacy, 21 st Ed., Lippincott, Williams & Wilkins (2005), the content of which is incorporated herein by reference.

術語“賦形劑”通常是指配製有效的藥物組合物所需要載體、稀釋劑和/或介質。 The term "excipient" generally refers to the carrier, diluent and/or medium required to formulate an effective pharmaceutical composition.

針對藥物或藥理學活性劑而言，術語“有效量”或“治療有效量”是指無毒的但能達到預期效果的藥物或藥劑的足夠用量。對於本發明中的口服劑型，組合物中一種活性物質的“有效量”是指與該組合物中另一種活性物質聯用時為了達到預期效果所需要的用量。有效量的確定因人而異，取决於受體的年齡和一般情况，也取决於具體的活性物質，個案中合適的有效量可以由本領域技術人員根據常規試驗確定。 For drugs or pharmacologically active agents, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a drug or agent that is non-toxic but can achieve the desired effect. For the oral dosage form of the present invention, the "effective amount" of one active substance in the composition refers to the amount required to achieve the desired effect when combined with another active substance in the composition. The determination of the effective amount varies from person to person, and depends on the age and general conditions of the recipient, as well as the specific active substance. The appropriate effective amount in a case can be determined by those skilled in the art according to routine experiments.

術語“活性成分”、“治療劑”，“活性物質”或“活性劑”是指一種化學實體，它可以有效地治療目標紊亂、疾病或病症。 The terms "active ingredient", "therapeutic agent", "active substance" or "active agent" refer to a chemical entity that can effectively treat the target disorder, disease or condition.

“任選”或“任選地”指的是隨後描述的事件或狀况可能但不是必需出現的，並且該描述包括其中所述事件或狀况發生的情况以及所述事件或狀况不發生的情况。 "Optional" or "optionally" means that the event or condition described later may but not necessarily occur, and the description includes the situation in which the event or condition occurs and the event or condition does not occur Case.

術語“被取代的”是指特定原子上的任意一個或多個氫原子被取代基取代，包括重氫和氫的變體，只要特定原子的價態是正常的並且取代後的化合物是穩定的。當取代基為酮基(即=O)時，意味著兩個氫原子被取代。酮取代不會發生在芳香基上。術語“任選被取代的”是指可以被取代，也可以不被取代，除非另有規定，取代基的種類和數目在化學上可以實現的基礎上可以是任意的。 The term "substituted" means that any one or more hydrogen atoms on a specific atom are replaced by substituents, including deuterium and hydrogen variants, as long as the valence of the specific atom is normal and the substituted compound is stable . When the substituent is a keto group (ie =O), it means that two hydrogen atoms are replaced. Ketone substitution does not occur on aromatic groups. The term "optionally substituted" means that it can be substituted or unsubstituted. Unless otherwise specified, the type and number of substituents can be arbitrary on the basis that they can be chemically realized.

當任何變量(例如R)在化合物的組成或結構中出現一次以上時，其在每一種情况下的定義都是獨立的。因此，例如，如果一個基團被0-2個R所取代，則所述基團可以任選地至多被兩個R所取代，並且每種情况下的R都有獨立的選項。此外，取代基和/或其變體的組合只有在這樣的組合會產生穩定的化合物的情况下才是被允許的。 When any variable (such as R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group can optionally be substituted with up to two Rs, and R has independent options in each case. In addition, combinations of substituents and/or variants thereof are only permitted if such combinations result in stable compounds.

當一個連接基團的數量為0時，比如-(CRR)0-，表示該連接基團為單鍵。 When the number of a linking group is 0, such as -(CRR) 0 -, it means that the linking group is a single bond.

當其中一個變量選自單鍵時，表示其連接的兩個基團直接相連，比如A-L-Z中L代表單鍵時表示該結構實際上是A-Z。 When one of the variables is selected from a single bond, it means that the two groups connected are directly connected. For example, when L in A-L-Z represents a single bond, it means that the structure is actually A-Z.

當一個取代基為空缺時，表示該取代基是不存在的，比如A-X中X為空缺時表示該結構實際上是A。 When a substituent is vacant, it means that the substituent is absent. For example, when X in A-X is vacant, it means that the structure is actually A.

當一個取代基的鍵可以交叉連接到一個環上的兩個原子時，這種取代基可以與這個環上的任意原子相鍵合。當所列舉的取代基中沒有指明其通過哪一個原子連接到化學結構通式中包括但未具體提及的化合物時，這種取代基可以通過其任何原子相鍵合。取代基和/或其變體的組合只有在 這樣的組合會產生穩定的化合物的情况下才是被允許的。例如，結構單元

Figure 105135042-A0305-02-0015-44
或
Figure 105135042-A0305-02-0015-43
表示其可在環己基或者環己二烯上的任意一個位 置發生取代。 When the bond of a substituent can be cross-connected to two atoms on a ring, the substituent can be bonded with any atom on the ring. When the listed substituents do not indicate through which atom they are connected to the compounds included in the general formula of the chemical structure but are not specifically mentioned, such substituents may be bonded through any of its atoms. Combinations of substituents and/or variants thereof are only permitted if such combinations result in stable compounds. For example, the structural unit
Figure 105135042-A0305-02-0015-44
or
Figure 105135042-A0305-02-0015-43
It means that it can be substituted at any position on the cyclohexyl or cyclohexadiene.

除非另有規定，術語“鹵代素”或“鹵素”本身或作為另一取代基的一部分表示氟、氯、溴或碘原子。此外，術語“鹵代烷基”意在包括單鹵代烷基和多鹵代烷基。例如，術語“鹵代(C1-C4)烷基”意在包括但不僅限於三氟甲基、2，2，2-三氟乙基、4-氯丁基和3-溴丙基等等。 Unless otherwise specified, the term "halogen" or "halogen" by itself or as part of another substituent means a fluorine, chlorine, bromine or iodine atom. In addition, the term "haloalkyl" is intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C 1 -C 4 )alkyl" is intended to include, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc. Wait.

鹵代烷基的實例包括但不僅限於：三氟甲基、三氯甲基、五氟乙基，和五氯乙基。“烷氧基”代表通過氧橋連接的具有特定數目碳原子的上述烷基。C1-6烷氧基包括C1、C2、C3、C4、C5和C6的烷氧基。烷氧基的例子包括但不限於：甲氧基、乙氧基、正丙氧基、異丙氧基、正丁氧基、二級丁氧基、三級丁氧基、正戊氧基和S-戊氧基。“環烷基”包括飽和環基，如環丙基、環丁基或環戊基。3-7環烷基包括C3、C4、C5、C6和C7環烷基。“鏈烯基”包括直鏈或支鏈構型的烴鏈，其中該鏈上任何的穩定位點上存在一個或多個碳-碳雙鍵，例如乙烯基和丙烯基。 Examples of haloalkyl groups include, but are not limited to: trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl. "Alkoxy" represents the above-mentioned alkyl group having a specified number of carbon atoms connected by an oxygen bridge. The C 1-6 alkoxy group includes C 1 , C 2 , C 3 , C 4 , C 5 and C 6 alkoxy groups. Examples of alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, secondary butoxy, tertiary butoxy, n-pentoxy and S -Pentyloxy. "Cycloalkyl" includes saturated cyclic groups such as cyclopropyl, cyclobutyl or cyclopentyl. 3-7 cycloalkyl groups include C 3 , C 4 , C 5 , C 6 and C 7 cycloalkyl groups. "Alkenyl" includes straight or branched hydrocarbon chains in which there are one or more carbon-carbon double bonds at any stable position on the chain, such as vinyl and propenyl.

術語“鹵”或“鹵素”是指氟、氯、溴和碘。 The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

除非另有規定，術語“雜”表示雜原子或雜原子團(即含有雜原子的原子團)，包括碳(C)和氫(H)以外的原子以及含有這些雜原子的原子團，例如包括氧(O)、氮(N)、硫(S)、矽(Si)、鍺(Ge)、鋁(Al)、硼(B)、-O-、-S-、=O、=S、-C(=O)O-、-C(=O)-、-C(=S)-、-S(=O)、-S(=O)2-，以及任選被取代的-C(=O)N(H)-、-N(H)-、-C(=NH)-、-S(=O)2N(H)-或-S(=O)N(H)-。 Unless otherwise specified, the term "hetero" means a heteroatom or group of atoms (ie, groups of atoms containing heteroatoms), including atoms other than carbon (C) and hydrogen (H) and groups of atoms containing these heteroatoms, such as oxygen (O ), nitrogen (N), sulfur (S), silicon (Si), germanium (Ge), aluminum (Al), boron (B), -O-, -S-, =O, =S, -C(= O)O-, -C(=O)-, -C(=S)-, -S(=O), -S(=O) 2 -, and optionally substituted -C(=O)N (H)-, -N(H)-, -C(=NH)-, -S(=O) 2 N(H)- or -S(=O)N(H)-.

除非另有規定，“環”表示被取代或未被取代的環烷基、雜環烷基、環烯基、雜環烯基、環炔基、雜環炔基、芳基或雜芳基。所謂的環包括單環、聯環、螺環、並環或橋環。環上原子的數目通常被定義為環的元數，例如，“5~7元環”是指環繞排列5~7個原子。除非另有規定，該環任選地包含1~3個雜原子。因此，“5~7元環”包括例如苯基吡啶和呱啶基；另一方面，術語“5~7元雜環烷基環”包括吡啶基和呱啶基，但不包括苯基。術語“環”還包括含有至少一個環的環系，其中的每一個“環”均獨立地符合上述定義。 Unless otherwise specified, "ring" means substituted or unsubstituted cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl, heterocycloalkynyl, aryl, or heteroaryl. The so-called ring includes a single ring, a bicyclic ring, a spiro ring, a parallel ring or a bridged ring. The number of atoms in the ring is usually defined as the number of ring members. For example, "5-7 membered ring" refers to the arrangement of 5-7 atoms around. Unless otherwise specified, the ring optionally contains 1 to 3 heteroatoms. Therefore, "5- to 7-membered ring" includes, for example, phenylpyridine and pyridyl; on the other hand, the term "5- to 7-membered heterocycloalkyl ring" includes pyridyl and pyridyl, but does not include phenyl. The term "ring" also includes a ring system containing at least one ring, where each "ring" independently meets the above definition.

除非另有規定，術語“雜環”或“雜環基”意指穩定的含雜原子或雜原子團的單環、雙環或三環，它們可以是飽和的、部分不飽和的或不飽和的(芳族的)，它們包含碳原子和1、2、3或4個獨立地選自N、O和S的環雜原子，其中上述任意雜環可以稠合到一個苯環上形成雙環。氮和硫雜原子可任選被氧化(即NO和S(O)p，p是1或2)。氮原子可以是被取代的或未取代的(即N或NR，其中R是H或本文已經定義過的其他取代基)。該雜環可以附著到任何雜原子或碳原子的側基上從而形成穩定的結構。如果產生的化合物是穩定的，本文所述的雜環可以發生碳位或氮位上的取代。雜環中的氮原子任選地被四級銨化。一個優選方案是，當雜環中S及O原子的總數超過1時，這些雜原子彼此不相鄰。另一個優選方案是，雜環中S及O原子的總數不超過1。如本文所用，術語“芳族雜環基團”或“雜芳基”意指穩定的5、6、7元單環或雙環或7、8、9或10元雙環雜環基的芳香環，它包含碳原子和1、2、3或4個獨立地選自N、O和S的環雜原子。氮原子可以是被取代的或未取代的(即N或NR，其中R是H或本文已經定義過的其他取代基)。氮和硫雜原子可任選被氧化(即NO和S(O)p,p是1或2)。值得注意的是，芳香雜環上S和O原子的總數不超過1。橋環也包含在雜環的定義中。 當一個或多個原子(即C、O、N或S)連接兩個不相鄰的碳原子或氮原子時形成橋環。優選的橋環包括但不限於：一個碳原子、兩個碳原子、一個氮原子、兩個氮原子和一個碳-氮基。值得注意的是，一個橋總是將單環轉換成三環。橋環中，環上的取代基也可以出現在橋上。 Unless otherwise specified, the term "heterocycle" or "heterocyclic group" means a stable monocyclic, bicyclic or tricyclic ring containing heteroatoms or heteroatoms, which may be saturated, partially unsaturated or unsaturated ( Aromatic), they contain carbon atoms and 1, 2, 3 or 4 ring heteroatoms independently selected from N, O and S, wherein any of the above heterocycles can be fused to a benzene ring to form a bicyclic ring. Nitrogen and sulfur heteroatoms can optionally be oxidized (ie NO and S(O)p, p is 1 or 2). The nitrogen atom may be substituted or unsubstituted (ie N or NR, where R is H or other substituents already defined herein). The heterocyclic ring can be attached to any heteroatom or carbon atom pendant group to form a stable structure. If the resulting compound is stable, the heterocyclic ring described herein can be substituted at the carbon or nitrogen position. The nitrogen atom in the heterocyclic ring is optionally quaternary ammonium. A preferred solution is that when the total number of S and O atoms in the heterocycle exceeds 1, these heteroatoms are not adjacent to each other. Another preferred solution is that the total number of S and O atoms in the heterocycle does not exceed 1. As used herein, the term "aromatic heterocyclic group" or "heteroaryl" means a stable 5-, 6, 7-membered monocyclic or bicyclic or 7, 8, 9 or 10-membered bicyclic heterocyclic aromatic ring, It contains carbon atoms and 1, 2, 3 or 4 ring heteroatoms independently selected from N, O and S. The nitrogen atom may be substituted or unsubstituted (ie N or NR, where R is H or other substituents already defined herein). Nitrogen and sulfur heteroatoms can optionally be oxidized (ie NO and S(O)p, p is 1 or 2). It is worth noting that the total number of S and O atoms in the aromatic heterocycle does not exceed 1. Bridged rings are also included in the definition of heterocycles. A bridged ring is formed when one or more atoms (ie, C, O, N, or S) connect two non-adjacent carbon atoms or nitrogen atoms. Preferred bridged rings include, but are not limited to: one carbon atom, two carbon atoms, one nitrogen atom, two nitrogen atoms and one carbon-nitrogen group. It is worth noting that a bridge always converts a single ring into a three ring. In bridged rings, substituents on the ring can also appear on the bridge.

雜環化合物的實例包括但不限於：吖啶基、吖辛因基、苯並咪唑基、苯並呋喃基、苯並巰基呋喃基、苯並巰基苯基、苯並噁唑基、苯並噁唑啉基、苯並噻唑基、苯並三唑基、苯並四唑基、苯並異噁唑基、苯並異噻唑基、苯並咪唑啉基、哢唑基、4aH-哢唑基、哢啉基、苯並二氫吡喃基、色烯、噌啉基十氫喹啉基、2H,6H-1,5,2-二噻嗪基、二氫呋喃並[2,3-b]四氫呋喃基、呋喃基、呋咱基、咪唑烷基、咪唑啉基、咪唑基、1H-吲唑基、吲哚烯基、二氫吲哚基、中氮茚基、吲哚基、3H-吲哚基、isatino基、異苯並呋喃基、異吲哚基、異二氫吲哚基、異喹啉基、異噻唑基、異噁唑基、亞甲二氧基苯基、嗎啉基、萘啶基，八氫異喹啉基、噁二唑基、1,2,3-噁二唑基、1,2,4-噁二唑基、1,2,5-噁二唑基、1,3,4-噁二唑基、噁唑烷基、噁唑基、羥吲哚基、嘧啶基、菲啶基、菲咯啉基、吩嗪、吩噻嗪、苯並黃嘌呤基、酚噁嗪基、酞嗪基、呱嗪基、呱啶基、呱啶酮基、4-呱啶酮基、胡椒基、蝶啶基、嘌呤基、吡喃基、吡嗪基、吡唑烷基、吡唑啉基、吡唑基、噠嗪基、吡啶並噁唑、吡啶並咪唑、吡啶並噻唑、吡啶基、吡咯烷基、吡咯啉基、2H-吡咯基、吡咯基、喹唑啉基、喹啉基、4H-喹嗪基、喹喔啉基、奎寧環基、四氫呋喃基、四氫異喹啉基、四氫喹啉基、四唑基，6H-1,2,5-噻二嗪基、1,2,3-噻二唑基、1,2,4-噻二唑基、1,2,5-噻二唑基、1,3,4-噻二唑基、噻蒽基、噻唑基、異噻唑基噻吩基、噻吩基、噻吩並噁唑基、噻吩並噻唑基、噻吩並咪唑基、三嗪基、1,2,3-三唑基、1,2,4-三唑基、1,2,5-三唑基、1,3,4-三唑基和呫噸基。還包括稠環和螺環化合物。 Examples of heterocyclic compounds include, but are not limited to: acridinyl, azeinyl, benzimidazolyl, benzofuranyl, benzomercaptofuranyl, benzomercaptophenyl, benzoxazolyl, benzoxanyl Azolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, oxazolyl, 4a H -oxazolyl , Cinolinyl, Chromanyl, Chromene, Cinolinyl Decahydroquinolinyl, 2 H ,6 H -1,5,2-Dithiazinyl, Dihydrofuro[2,3 -b ] Tetrahydrofuryl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1 H -indazolyl, indolenyl, indoline, indolyl, indolyl , 3 H -indolyl, isatino, isobenzofuranyl, isoindolyl, isoindolinyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl , Morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxa Diazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindole, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazine, phenothiazine, benzo Xanthinyl, phenoxazinyl, phthalazinyl, pyrazinyl, pyridinyl, pyridinonyl, 4-pyridinyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl , Pyrazolidinyl, pyrazoline, pyrazolyl, pyridazinyl, pyridoxazole, pyridoimidazole, pyridothiazole, pyridyl, pyrrolidinyl, pyrrolinyl, 2 H -pyrrolyl, pyrrole Group, quinazolinyl, quinolinyl, 4 H -quinazinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6 H -1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4 -Thiadiazolyl, thiaanthryl, thiazolyl, isothiazolylthienyl, thienyl, thienooxazolyl, thienothiazolyl, thienoimidazolyl, triazinyl, 1,2,3-triazole 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthene. Also includes fused ring and spiro ring compounds.

除非另有規定，術語“烴基”或者其下位概念(比如烷基、烯基、炔基、芳基等等)本身或者作為另一取代基的一部分表示直鏈的、支鏈的或環狀的烴原子團或其組合，可以是完全飽和的(如烷基)、單元或多元不飽和的(如烯基、炔基、芳基)，可以是單取代或多取代的，可以是一價(如甲基)、二價(如亞甲基)或者多價(如次甲基)，可以包括二價或多價原子團，具有指定數量的碳原子(如C1-C12表示1至12個碳，C1-12選自C1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11和C12；C3-12選自C3、C4、C5、C6、C7、C8、C9、C10、C11和C12。)。“烴基”包括但不限於脂肪烴基和芳香烴基，所述脂肪烴基包括鏈狀和環狀，具體包括但不限於烷基、烯基、炔基，所述芳香烴基包括但不限於6-12元的芳香烴基，例如苯、萘等。在一些實施例中，術語“烴基”表示直鏈的或支鏈的原子團或它們的組合，可以是完全飽和的、單元或多元不飽和的，可以包括二價和多價原子團。飽和烴原子團的實例包括但不限於甲基、乙基、正丙基、異丙基、正丁基、三級丁基、異丁基、二級丁基、異丁基、環己基、(環己基)甲基、環丙基甲基，以及正戊基、正己基、正庚基、正辛基等原子團的同系物或異構體。不飽和烴基具有一個或多個雙鍵或三鍵，其實例包括但不限於乙烯基、2-丙烯基、丁烯基、巴豆基、2-異戊烯基、2-(丁二烯基)、2，4-戊二烯基、3-(1，4-戊二烯基)、乙炔基、1-和3-丙炔基，3-丁炔基，以及更高級的同系物和異構體。 Unless otherwise specified, the term "hydrocarbyl" or its subordinate concepts (such as alkyl, alkenyl, alkynyl, aryl, etc.) by itself or as part of another substituent means linear, branched or cyclic Hydrocarbon radicals or combinations thereof, can be fully saturated (such as alkyl), unit or polyunsaturated (such as alkenyl, alkynyl, aryl), can be mono- or multi-substituted, and can be monovalent (such as Methyl), divalent (such as methylene) or multivalent (such as methine), can include divalent or multivalent atom groups, with a specified number of carbon atoms (such as C 1 -C 12 represents 1 to 12 carbon , C 1-12 is selected from C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 and C 12 ; C 3-12 is selected from C 3. C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 and C 12 .). "Hydrocarbon group" includes but is not limited to aliphatic hydrocarbon groups and aromatic hydrocarbon groups. The aliphatic hydrocarbon groups include chain and cyclic, specifically including but not limited to alkyl groups, alkenyl groups, and alkynyl groups. The aromatic hydrocarbon groups include but are not limited to 6-12 members. The aromatic hydrocarbon group, such as benzene, naphthalene, etc. In some embodiments, the term "hydrocarbyl" refers to linear or branched atomic groups or combinations thereof, which can be fully saturated, unitary or polyunsaturated, and can include divalent and multivalent atomic groups. Examples of saturated hydrocarbon radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tertiary butyl, isobutyl, secondary butyl, isobutyl, cyclohexyl, (cyclic Hexyl) methyl, cyclopropylmethyl, and homologs or isomers of atomic groups such as n-pentyl, n-hexyl, n-heptyl, and n-octyl. Unsaturated hydrocarbon groups have one or more double bonds or triple bonds, examples of which include but are not limited to vinyl, 2-propenyl, butenyl, crotyl, 2-isopentenyl, 2-(butadienyl) , 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers body.

除非另有規定，術語“雜烴基”或者其下位概念(比如雜烷基、雜烯基、雜炔基、雜芳基等等)本身或者與另一術語聯合表示穩定的直鏈的、支鏈的或環狀的烴原子團或其組合，有一定數目的碳原子和至少一個雜原子組成。在一些實施例中，術語“雜烴基”本身或者與另一術語聯合表示穩定的直鏈的、支鏈的烴原子團或其組合物，有一定數目的碳原子和至少一 個雜原子組成。在一個典型實施例中，雜原子選自B、O、N和S，其中氮和硫原子任選地被氧化，氮雜原子任選地被四級銨化。雜原子B、O、N和S可以位於雜烴基的任何內部位置(包括該烴基附著於分子其餘部分的位置)。實例包括但不限於-CH2-CH2-O-CH3、-CH2-CH2-NH-CH3、-CH2-CH2-N(CH3)-CH3、-CH2-S-CH2-CH3、-CH2-CH2、-S(O)-CH3、-CH2-CH2-S(O)2-CH3、-CH=CH-O-CH3、-CH2-CH=N-OCH3和-CH=CH-N(CH3)-CH3。至多兩個雜原子可以是連續的，例如-CH2-NH-OCH3。 Unless otherwise specified, the term "heterohydrocarbyl" or its subordinate concepts (such as heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, etc.) by itself or in combination with another term means a stable linear, branched chain Cyclic or cyclic hydrocarbon radicals or combinations thereof, consisting of a certain number of carbon atoms and at least one heteroatom. In some embodiments, the term "heterohydrocarbyl" by itself or in combination with another term means a stable linear or branched hydrocarbon radical or a combination thereof, consisting of a certain number of carbon atoms and at least one heteroatom. In a typical embodiment, the heteroatom is selected from B, O, N, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. The heteroatoms B, O, N, and S can be located in any internal position of the heterohydrocarbyl group (including the position where the hydrocarbyl group is attached to the rest of the molecule). Examples include but are not limited to -CH 2 -CH 2 -O-CH 3 , -CH 2 -CH 2 -NH-CH 3 , -CH 2 -CH 2 -N(CH 3 )-CH 3 , -CH 2 -S -CH 2 -CH 3 , -CH 2 -CH 2 , -S(O)-CH 3 , -CH 2 -CH 2 -S(O) 2 -CH 3 , -CH=CH-O-CH 3 ,- CH 2 -CH=N-OCH 3 and -CH=CH-N(CH 3 )-CH 3 . Up to two heteroatoms can be continuous, for example -CH 2 -NH-OCH 3 .

術語“烷氧基”、“烷氨基”和“烷硫基”(或硫代烷氧基)屬慣用表達，是指分別通過一個氧原子、氨基或硫原子連接到分子的其餘部分的那些烷基基團。 The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are customary expressions and refer to those alkyl groups attached to the rest of the molecule through an oxygen atom, an amino group, or a sulfur atom, respectively. Group.

除非另有規定，術語“烷基”用於表示直鏈或支鏈的飽和烴基，可以是單取代(如-CH2F)或多取代的(如-CF3)，可以是一價(如甲基)、二價(如亞甲基)或者多價(如次甲基)。烷基的例子包括甲基(Me)，乙基(Et)，丙基(如，n-丙基和異丙基)，丁基(如，n-丁基，異丁基，s-丁基，t-丁基)，戊基(如，n-戊基，異戊基，新戊基)等。 Unless otherwise specified, the term "alkyl" is used to denote a linear or branched saturated hydrocarbon group, which may be mono-substituted (such as -CH 2 F) or multi-substituted (such as -CF 3 ), and may be monovalent (such as Methyl), divalent (such as methylene) or multivalent (such as methine). Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, s-butyl) , T-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl) and the like.

除非另有規定，術語“鹵代素”或“鹵素”本身或作為另一取代基的一部分表示氟、氯、溴或碘原子。此外，術語“鹵代烷基”意在包括單鹵代烷基和多鹵代烷基。例如，術語“鹵代(C1-C4)烷基”意在包括但不僅限於三氟甲基、2，2，2-三氟乙基、4-氯丁基和3-溴丙基等等。除非另有規定，鹵代烷基的實例包括但不僅限於：三氟甲基、三氯甲基、五氟乙基，和五氯乙基。 Unless otherwise specified, the term "halogen" or "halogen" by itself or as part of another substituent means a fluorine, chlorine, bromine or iodine atom. In addition, the term "haloalkyl" is intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C 1 -C 4 )alkyl" is intended to include, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc. Wait. Unless otherwise specified, examples of haloalkyl include, but are not limited to: trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl.

“烷氧基”代表通過氧橋連接的具有特定數目碳原子的上述烷基，除非另有規定，C1-6烷氧基包括C1、C2、C3、C4、C5和C6的烷氧基。烷氧基 的例子包括但不限於：甲氧基、乙氧基、正丙氧基、異丙氧基、正丁氧基、二級丁氧基、三級丁氧基、正戊氧基和S-戊氧基。 "Alkoxy" represents the above-mentioned alkyl group having a specified number of carbon atoms connected by an oxygen bridge. Unless otherwise specified, C 1-6 alkoxy includes C 1 , C 2 , C 3 , C 4 , C 5 and C 6 's alkoxy group. Examples of alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, secondary butoxy, tertiary butoxy, n-pentoxy and S -Pentyloxy.

除非另有規定，術語“環烴基”、“雜環烴基”或者其下位概念(比如芳基、雜芳基、環烷基、雜環烷基、環烯基、雜環烯基、環炔基、雜環炔基等等)本身或與其他術語聯合分別表示環化的“烴基”、“雜烴基”。此外，就雜烴基或雜環烴基(比如雜烷基、雜環烷基)而言，雜原子可以占據該雜環附著於分子其餘部分的位置。環烷基的實例包括但不限於環戊基、環己基、1-環己烯基、3-環己烯基、環庚基等。雜環基的非限制性實例包括1-(1,2,5,6-四氫吡啶基)、1-呱啶基、2-呱啶基，3-呱啶基、4-嗎啉基、3-嗎啉基、四氫呋喃-2-基、四氫呋喃吲哚-3-基、四氫噻吩-2-基、四氫噻吩-3-基，1-呱嗪基和2-呱嗪基。 Unless otherwise specified, the term "cycloalkyl", "heterocycloalkyl" or its subordinate concepts (such as aryl, heteroaryl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl , Heterocycloalkynyl, etc.) by itself or in combination with other terms respectively represent cyclized "hydrocarbyl" and "heterohydrocarbyl". In addition, in the case of a heterohydrocarbyl group or a heterocyclic hydrocarbyl group (such as heteroalkyl, heterocycloalkyl), a heteroatom may occupy the position where the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Non-limiting examples of heterocyclic groups include 1-(1,2,5,6-tetrahydropyridyl), 1-pyridinyl, 2-pyridinyl, 3-pyridinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuranindol-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl and 2-piperazinyl.

除非另有規定，術語“芳基”表示多不飽和的芳族烴取代基，可以是單取代、二取代或多取代的，可以是一價、二價或者多價，它可以是單環或多環(優選1至3個環)，它們稠合在一起或共價連接。術語“雜芳基”是指含有一至四個雜原子的芳基(或環)。在一個示範性實例中，雜原子選自B、N、O和S，其中氮和硫原子任選地被氧化，氮原子任選地被四級銨化。雜芳基可通過雜原子連接到分子的其餘部分。芳基或雜芳基的非限制性實施例包括苯基、1-萘基、2-萘基、4-聯苯基、1-吡咯基、2-吡咯基、3-吡咯基、3-吡唑基、2-咪唑基、4-咪唑基、吡嗪基、2-噁唑基、4-噁唑基、2-苯基-4-噁唑基、5-噁唑基、3-異噁唑基、4-異噁唑基、5-異噁唑基、2-噻唑基、4-噻唑基、5-噻唑基、2-呋喃基、3-呋喃基、2-噻吩基、3-噻吩基、2-吡啶基、3-吡啶基、4-吡啶基、2-嘧啶基、4-嘧啶基、5-苯並噻唑基、嘌呤基、2-苯並咪唑基、5-吲哚基、1-異喹啉基、5-異喹啉基、2-喹喔啉基、 5-喹喔啉基、3-喹啉基和6-喹啉基。上述任意一個芳基和雜芳基環系的取代基選自下文所述的可接受的取代基。 Unless otherwise specified, the term "aryl" means a polyunsaturated aromatic hydrocarbon substituent, which can be mono-, di- or poly-substituted, monovalent, divalent or polyvalent, and it can be monocyclic or Multiple rings (preferably 1 to 3 rings), which are fused together or linked covalently. The term "heteroaryl" refers to an aryl group (or ring) containing one to four heteroatoms. In an exemplary example, the heteroatom is selected from B, N, O, and S, wherein nitrogen and sulfur atoms are optionally oxidized, and nitrogen atoms are optionally quaternary ammonium. Heteroaryl groups can be attached to the rest of the molecule through heteroatoms. Non-limiting examples of aryl or heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrrolyl Azolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxyl Azolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thiophene Group, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolinyl, 5-isoquinolinyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolinyl and 6-quinolinyl. The substituents of any one of the above-mentioned aryl and heteroaryl ring systems are selected from the acceptable substituents described below.

為簡便起見，芳基在與其他術語聯合使用時(例如芳氧基、芳硫基、芳烷基)包括如上定義的芳基和雜芳基環。因此，術語“芳烷基”意在包括芳基附著於烷基的那些原子團(例如苄基、苯乙基、吡啶基甲基等)，包括其中碳原子(如亞甲基)已經被例如氧原子代替的那些烷基，例如苯氧基甲基、2-吡啶氧甲基3-(1-萘氧基)丙基等。 For brevity, aryl when used in conjunction with other terms (e.g., aryloxy, arylthio, aralkyl) includes aryl and heteroaryl rings as defined above. Therefore, the term "aralkyl" is meant to include those atomic groups where an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, etc.), including where the carbon atom (e.g., methylene) has been replaced by, for example, oxygen Those alkyl groups substituted by atoms, for example, phenoxymethyl, 2-pyridyloxymethyl 3-(1-naphthyloxy)propyl and the like.

術語“離去基團”是指可以被另一種官能團或原子通過取代反應(例如親和取代反應)所取代的官能團或原子。例如，代表性的離去基團包括三氟甲磺酸酯；氯、溴、碘；磺酸酯基，如甲磺酸酯、甲苯磺酸酯、對溴苯磺酸酯、對甲苯磺酸酯等；醯氧基，如乙醯氧基、三氟乙醯氧基等等。 The term "leaving group" refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (for example, an affinity substitution reaction). For example, representative leaving groups include triflate; chlorine, bromine, iodine; sulfonate groups, such as mesylate, tosylate, p-bromobenzenesulfonate, p-toluenesulfonic acid Esters, etc.; acetoxy groups, such as acetoxy, trifluoroacetoxy and the like.

術語“保護基”包括但不限於“氨基保護基”、“羥基保護基”或“巰基保護基”。術語“氨基保護基”是指適合用於阻止氨基氮位上副反應的保護基團。代表性的氨基保護基包括但不限於：甲醯基；醯基，例如鏈烷醯基(如乙醯基、三氯乙醯基或三氟乙醯基)；烷氧基羰基，如三級丁氧基羰基(Boc)；芳基甲氧羰基，如苄氧羰基(Cbz)和9-芴甲氧羰基(Fmoc)；芳基甲基，如苄基(Bn)、三苯甲基(Tr)、1,1-二-(4'-甲氧基苯基)甲基；甲矽烷基，如三甲基甲矽烷基(TMS)和三級丁基二甲基甲矽烷基(TBS)等等。術語“羥基保護基”是指適合用於阻止羥基副反應的保護基。代表性羥基保護基包括但不限於：烷基，如甲基、乙基和三級丁基；醯基，例如鏈烷醯基(如乙醯基)；芳基甲基，如苄基(Bn)，對甲氧基苄基(PMB)、9-芴基甲基(Fm)和二苯基甲基(二苯甲基，DPM)；甲矽烷基，如三甲基甲矽烷基(TMS)和三級丁基二甲基甲矽烷基(TBS)等等。 The term "protecting group" includes, but is not limited to, "amino protecting group", "hydroxy protecting group" or "thiol protecting group". The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the amino nitrogen position. Representative amino protecting groups include, but are not limited to: methanoyl; anolyte, such as alkanoyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl, such as tertiary Butoxycarbonyl (Boc); arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethyloxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr ), 1,1-bis-(4'-methoxyphenyl) methyl; silyl groups, such as trimethylsilyl (TMS) and tertiary butyldimethylsilyl (TBS), etc. Wait. The term "hydroxyl protecting group" refers to a protecting group suitable for preventing side reactions of the hydroxyl group. Representative hydroxy protecting groups include, but are not limited to: alkyl groups, such as methyl, ethyl, and tertiary butyl; acyl groups, such as alkanoyl groups (such as acetyl); arylmethyl groups, such as benzyl (Bn ), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups, such as trimethylsilyl (TMS) And tertiary butyldimethylsilyl (TBS) and so on.

本發明的化合物可以通過本領域技術人員所熟知的多種合成方法來製備，包括下面列舉的具體實施方式、其與其他化學合成方法的結合所形成的實施方式以及本領域技術上人員所熟知的等同替換方式，優選的實施方式包括但不限於本發明的實施例。 The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and equivalents well known to those skilled in the art. Alternatively, preferred implementations include, but are not limited to, the embodiments of the present invention.

本發明所使用的所有溶劑是市售的，無需進一步純化即可使用。本發明採用下述縮略詞：化合物1為實施例51異構體2；Pen.為己酮可可鹼；INT-747為6-乙基鵝去氧膽酸；aq代表水；HATU代表O-(7-氮雜苯並三唑-1-基)-N，N，N'，N'-四甲基脲六氟磷酸鹽；EDC代表N-(3-二甲基氨基丙基)-N'-乙基碳二亞胺鹽酸鹽；m-CPBA代表3-氯過氧苯甲酸；eq代表當量、等量；CDI代表羰基二咪唑；DCM代表二氯甲烷；PE代表石油醚；DIAD代表偶氮二羧酸二異丙酯；DMF代表N，N-二甲基甲醯胺；DMSO代表二甲亞碸；EtOAc代表乙酸乙酯；EtOH代表乙醇；MeOH代表甲醇；CBz代表苄氧羰基，是一種胺保護基團；BOC代表三級丁基羰基是一種胺保護基團；HOAc代表乙酸；NaCNBH3代表氰基硼氫化鈉；r.t.代表室溫；O/N代表過夜；THF代表四氫呋喃；Boc2O代表二-三級丁基二碳酸酯；TFA代表三氟乙酸；DIPEA代表二異丙基乙基胺；SOCl2代表氯化亞碸；CS2代表二硫化碳；TsOH代表對甲苯磺酸；NFSI代表N-氟-N-(苯磺醯基)苯磺醯胺；NCS代表1-氯吡咯烷-2,5-二酮；n-Bu4NF代表氟化四丁基銨；iPrOH代表2-丙醇；mp代表熔點；LDA代表二異丙基胺基鋰；TMSCF3代表三氟甲基三甲基矽烷；Ti(Oi-Pr)4代表鈦酸四異丙酯；MSCl代表甲烷磺醯氯；DMAP代表N,N-二甲基-4-氨基吡啶；TEA代表三乙胺；BnBr代表苄溴；DIEA代表二異丙基乙胺；BH3DMS代表硼烷二甲硫醚；DMP代表戴斯馬丁過碘烷；TBAF代表四丁基氟化胺；HOBT代表1-羥基苯並三唑；AIBN代表偶氮二異丁腈；NBS 代表N-溴代丁二醯亞胺；RT緩衝液代表逆轉錄緩衝液；dNTP代表脫氧核糖核苷三磷酸；PBS代表磷酸鹽緩衝液。 All solvents used in the present invention are commercially available and can be used without further purification. The present invention uses the following acronyms: Compound 1 is Example 51 Isomer 2; Pen. is Pentoxifylline; INT-747 is 6-ethylchenodeoxycholic acid; aq represents water; HATU represents O- (7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate; EDC stands for N-(3-dimethylaminopropyl)-N '-Ethylcarbodiimide hydrochloride; m-CPBA stands for 3-chloroperoxybenzoic acid; eq stands for equivalent, equivalent; CDI stands for carbonyl diimidazole; DCM stands for dichloromethane; PE stands for petroleum ether; DIAD stands for Diisopropyl azodicarboxylate; DMF stands for N,N-dimethylformamide; DMSO stands for dimethyl sulfide; EtOAc stands for ethyl acetate; EtOH stands for ethanol; MeOH stands for methanol; CBz stands for benzyloxycarbonyl, Is an amine protecting group; BOC stands for tertiary butylcarbonyl is an amine protecting group; HOAc stands for acetic acid; NaCNBH 3 stands for sodium cyanoborohydride; rt stands for room temperature; O/N stands for overnight; THF stands for tetrahydrofuran; Boc 2 O stands for di-tertiary butyl dicarbonate; TFA stands for trifluoroacetic acid; DIPEA stands for diisopropylethylamine; SOCl 2 stands for sulphurous chloride; CS 2 stands for carbon disulfide; TsOH stands for p-toluenesulfonic acid; NFSI Represents N-fluoro-N-(benzenesulfonyl)benzenesulfonamide; NCS represents 1-chloropyrrolidine-2,5-dione; n -Bu 4 NF represents tetrabutylammonium fluoride; iPrOH represents 2- Propanol; mp stands for melting point; LDA stands for lithium diisopropylamide; TMSCF 3 stands for trifluoromethyltrimethylsilane; Ti(Oi-Pr) 4 stands for tetraisopropyl titanate; MSCl stands for methanesulfonyl chloride ; DMAP stands for N,N-dimethyl-4-aminopyridine; TEA stands for triethylamine; BnBr stands for benzyl bromide; DIEA stands for diisopropylethylamine; BH 3 DMS stands for borane dimethyl sulfide; DMP stands for Dai Martin periodane; TBAF stands for tetrabutylamine fluoride; HOBT stands for 1-hydroxybenzotriazole; AIBN stands for azobisisobutyronitrile; NBS stands for N-bromosuccinimide; RT buffer stands for Reverse transcription buffer; dNTP stands for deoxyribonucleoside triphosphate; PBS stands for phosphate buffer.

化合物經手工或者ChemDraw®軟件命名，市售化合物采用供應商目錄名稱。 Compounds are named manually or by ChemDraw® software, and commercially available compounds use supplier catalog names.

圖1：MCD小鼠肝臟HE染色圖片；圖2：MCD小鼠肝臟組織學評分，數據表示為均值±標準誤，n=8，####p<0.0001,###p<0.001 vs.MCD對照飲食+溶媒對照組，***p<0.001 vs.MCD飲食+溶媒對照組，單因素重複方差分析及Tukey’s組間兩兩比較；圖3：化合物1對MCD小鼠肝臟TG水平的影響，數據表示為均值±標準誤，n=7~8。####p<0.0001 vs.MCD對照飲食+溶媒對照組。單因素重複方差分析及Tukey’s組間兩兩比較，溢出值(>均值+2×標準差，或<均值-2×標準差)已去除；圖4：化合物1對MCD小鼠肝臟TNF-αmRNA水平的影響。數據表示為均值±標準誤，n=7~8。#p<0.05 vs.MCD對照飲食+溶媒對照組。單因素重複方差分析及Tukey’s組間兩兩比較，溢出值(>均值+2×標準差，或<均值-2×標準差)已去除；圖5：化合物1抑制MCD小鼠肝臟IL-1β水平，數據表示為均值±標準誤，n=7~8。###p<0.001 vs.MCD對照飲食+溶媒對照組，*p<0.001 vs.MCD飲食+溶媒對照組。單因素重複方差分析及Tukey’s組間兩兩比較，溢出值(>均值+2×標準差，或<均值-2×標準差)已去除； 圖6：化合物1抑制MCD小鼠血漿TNF-α和IL-1β水平，數據表示為均值±標準誤，n=7~8。####p<0.0001 vs.MCD對照飲食+溶媒對照組。*p<0.05,**p<0.01 vs.MCD飲食+溶媒對照組，單因素重複方差分析及Uncorrected Fisher’s LSD組間兩兩比較，溢出值(>均值+2×標準差，或<均值-2×標準差)已去除；圖7：化合物1對MCD小鼠血漿ALT與AST水平的影響，數據表示為均值±標準誤，n=7~8。####p<0.0001 vs.MCD對照飲食+溶媒對照組。單因素重複方差分析及Tukey’s組間兩兩比較，溢出值(>均值+2×標準差，或<均值-2×標準差)已去除；圖8：nSTZ+HFD小鼠肝臟HE染色圖片：A.對照組(STZ溶劑對照+正常飲食)B.模型組(STZ+高脂飲食)+溶媒對照(t.i.d.)；C.模型組(STZ+高脂飲食)+己酮可可鹼(100mg/kg,p.o,t.i.d.)；以及D.模型組(STZ+高脂飲食)+化合物1(30mg/kg,p.o,b.i.d.)；圖9：nSTZ+HFD小鼠肝臟組織學NAS評分。數據表示為均值±標準誤，n=12~17。####p<0.0001,##p<0.01 vs.對照組。單因素重複方差分析及Uncorrected Fish’s LSD組間兩兩比較；圖10：STZ+HFD小鼠肝臟天狼星紅染色圖片；圖11：nSTZ+HFD小鼠肝臟纖維化評分。數據表示為均值±標準誤，n=3，##p<0.01 vs.對照組，*p<0.05 vs.模型組+溶媒對照。單因素重複方差分析及Uncorrected Fish’s LSD組間兩兩比較；圖12：化合物1對nSTZ+HFD小鼠肝臟生物標志物mRNA水平的影響。數據表示為均值±標準誤，n=4~20。#p<0.05,##p<0.01 vs.對照組，*p<0.05 vs.模 型組+溶媒對照。單因素重複方差分析及Uncorrected Fish’s LSD組間兩兩比較，溢出值(>均值+2×標準差，或<均值-2×標準差)已去除；圖13：化合物1對nSTZ+HFD小鼠血清生化指標的影響。數據表示為均值±標準誤，n=15~17。###p<0.001，####p<0.0001 vs.對照組。單因素重複方差分析及Uncorrected Fish’s LSD組間兩兩比較，溢出值(>均值+2×標準差，或<均值-2×標準差)已去除；圖14：膽汁淤積型肝纖維化模型大鼠肝臟HE染色圖片，100倍放大。 Figure 1: HE staining image of liver in MCD mice; Figure 2: Histological score of liver in MCD mice. Data are expressed as mean ± standard error, n=8, ####p<0.0001, ###p<0.001 vs. MCD control diet + vehicle control group, ***p<0.001 vs. MCD diet + vehicle control group, single factor repeated analysis of variance and pairwise comparison between Tukey's groups; Figure 3: Effect of compound 1 on liver TG levels in MCD mice , Data is expressed as mean ± standard error, n=7~8. ####p<0.0001 vs. MCD control diet + vehicle control group. One-way repeated analysis of variance and pairwise comparisons between Tukey's groups, the overflow value (>mean + 2×standard deviation, or <mean-2×standard deviation) has been removed; Figure 4: Compound 1 on the liver TNF-αmRNA levels of MCD mice Impact. Data are expressed as mean ± standard error, n=7~8. #p<0.05 vs. MCD control diet + vehicle control group. One-way repeated analysis of variance and pairwise comparisons between Tukey's groups, the overflow value (>mean+2×standard deviation, or <mean-2×standard deviation) has been removed; Figure 5: Compound 1 inhibits liver IL-1β levels in MCD mice , Data is expressed as mean ± standard error, n=7~8. ###p<0.001 vs. MCD control diet + vehicle control group, *p<0.001 vs. MCD diet + vehicle control group. Single-factor repeated analysis of variance and pairwise comparisons between Tukey's groups, the overflow value (>mean+2×standard deviation, or <mean-2×standard deviation) has been removed; Figure 6: Compound 1 inhibits plasma TNF-α and TNF-α in MCD mice IL-1β level, data are expressed as mean ± standard error, n=7~8. ####p<0.0001 vs. MCD control diet + vehicle control group. *p<0.05, **p<0.01 vs. MCD diet + vehicle control group, one-way repeated analysis of variance and pairwise comparisons between Uncorrected Fisher's LSD groups, overflow (>mean+2×standard deviation, or <mean-2) ×standard deviation) has been removed; Figure 7: the effect of compound 1 on plasma ALT and AST levels in MCD mice, data are expressed as mean±standard error, n=7~8. ####p<0.0001 vs. MCD control diet + vehicle control group. One-way repeated analysis of variance and pairwise comparisons between Tukey's groups, the overflow value (>mean+2×standard deviation, or <mean-2×standard deviation) has been removed; Figure 8: HE staining picture of nSTZ+HFD mouse liver: A Control group (STZ solvent control + normal diet) B. Model group (STZ + high fat diet) + vehicle control (tid); C. Model group (STZ + high fat diet) + pentoxifylline (100mg/kg, po, tid); and D. Model group (STZ+high-fat diet)+Compound 1 (30mg/kg, po, bid); Figure 9: nSTZ+HFD mouse liver histology NAS score. Data are expressed as mean ± standard error, n=12~17. ####p<0.0001,##p<0.01 vs. the control group. Single factor repeated analysis of variance and pairwise comparison between Uncorrected Fish's LSD groups; Figure 10: Sirius red staining image of STZ+HFD mouse liver; Figure 11: Liver fibrosis score of nSTZ+HFD mouse. Data are expressed as mean ± standard error, n=3, ##p<0.01 vs. control group, *p<0.05 vs. model group + vehicle control. Single factor repeated analysis of variance and pairwise comparisons between Uncorrected Fish's LSD groups; Figure 12: The effect of compound 1 on the mRNA level of liver biomarkers in nSTZ+HFD mice. Data are expressed as mean ± standard error, n=4-20. #p <0.05, ##p <0.01 vs. control group, * p <0.05 vs. model group + vehicle control. Single factor repeated analysis of variance and pairwise comparisons between Uncorrected Fish's LSD groups, overflow values (>mean + 2×standard deviation, or <mean-2×standard deviation) have been removed; Figure 13: Compound 1 vs. nSTZ+HFD mouse serum The impact of biochemical indicators. Data are expressed as mean ± standard error, n=15-17. ###p<0.001, ####p<0.0001 vs. the control group. One-way repeated analysis of variance and pairwise comparisons between Uncorrected Fish's LSD groups, the overflow value (>mean+2×standard deviation, or <mean-2×standard deviation) has been removed; Figure 14: cholestatic liver fibrosis model rats HE stained image of liver, magnified 100 times.

A：組-1，健康對照；B：組-2，模型對照；C：組-3 INT-747-20mg/kg，QD；D：組-4，己酮可可鹼-100mg/kg，TID；E：組-5，化合物1-1mg/kg，BID；F：組-6，化合物1-3mg/kg，BID；G：組-7，化合物1-10mg/kg，BID。 A: Group-1, healthy control; B: Group-2, model control; C: Group-3 INT-747-20mg/kg, QD; D: Group-4, Pentoxifylline-100mg/kg, TID; E: group-5, compound 1-1 mg/kg, BID; F: group-6, compound 1-3 mg/kg, BID; G: group-7, compound 1-10 mg/kg, BID.

圖15：膽汁淤積型肝纖維化模型大鼠肝臟病理炎症評分。數據以均值±SEM標示，n=12。***p<0.001vs模型組；圖16：膽汁淤積型肝纖維化模型大鼠肝臟天狼星紅染色圖片，50倍放大。 Figure 15: Score of liver pathological inflammation in cholestatic liver fibrosis model rats. Data are indicated as mean±SEM, n=12. ***p<0.001 vs model group; Figure 16: Sirius red staining image of liver in a cholestatic liver fibrosis model rat, 50 times magnification.

A：組-1，健康對照；B：組-2，模型對照；C：組-3 INT-747-20mg/kg，QD；D：組-4，己酮可可鹼-100MG/kg，TID；E：組-5，化合物1-1mg/kg，BID；F：組-6，化合物1-3mg/kg，BID；G：組-7，化合物1-10mg/kg，BID。 A: Group-1, healthy control; B: Group-2, model control; C: Group-3 INT-747-20mg/kg, QD; D: Group-4, Pentoxifylline-100MG/kg, TID; E: group-5, compound 1-1 mg/kg, BID; F: group-6, compound 1-3 mg/kg, BID; G: group-7, compound 1-10 mg/kg, BID.

圖17：膽汁淤積型肝纖維化模型大鼠肝臟纖維化面積。數據以均值±SEM標示，n=12，與Vechile control組作比較，*p<0.05，***p<0.001；圖18：CCl4誘導的小鼠肝纖維化模型小鼠肝臟HE染色圖片，100倍放大。 Figure 17: Liver fibrosis area of cholestatic liver fibrosis model rat. Data are indicated by mean±SEM, n=12, compared with Vechile control group, *p<0.05, ***p<0.001; Figure 18: CCl 4 induced mouse liver fibrosis model mouse liver HE staining picture, 100 times magnification.

A：組-1，健康對照；B：組-2，模型對照；C：組-3 INT-747-20mg/kg，QD；D：組-4，己酮可可鹼-100mg/kg，TID；E：組-5，化合物1-1mg/kg，BID；F：組-6，化合物1-3mg/kg，BID；G：組-7，化合物1-10mg/kg，BID； 圖19：CCl4誘導的小鼠肝纖維化模型小鼠肝臟病理炎症評分。數據以均值±SEM標示，n=15。與模型組作比較，***p<0.001,**p<0.01,*p<0.05；圖20：CCl4誘導的小鼠肝纖維化模型小鼠肝臟氣球樣變分值。數據以均值±SEM標示，n=15，與模型組作比較，***p<0.001,**p<0.01,*p<0.05；圖21：CCl4誘導的小鼠肝纖維化模型小鼠肝臟水樣變性分值。數據以均值±SEM標示，n=15，與模型組作比較，***p<0.001,**p<0.01,*p<0.05；圖22：CCl4誘導的小鼠肝纖維化模型小鼠肝臟壞死分值。數據以均值±SEM標示，n=15，與模型組作比較，***p<0.001,**p<0.01,*p<0.05；圖23：CCl4誘導的小鼠肝纖維化模型小鼠肝臟損傷綜合分值。數據以均值±SEM標示，n=15，與模型組作比較，***p<0.001,**p<0.01,*p<0.05；圖24：CCl4誘導的小鼠肝纖維化模型小鼠肝臟天狼星紅染色圖片，50倍放大：A：組-1，健康對照；B：組-2，模型對照；C：組-3 INT-747-20mg/kg,QD；D：組-4，己酮可可鹼-100mg/kg，TID；E：組-5，化合物1-1mg/kg，BID；F：組-6，化合物1-3mg/kg，BID；G：組-7，化合物1-10mg/kg，BID；以及圖25：CCl4誘導的小鼠肝纖維化模型小鼠肝臟纖維化面積。數據以均值±SEM標示，n=15，與模型組作比較，*p<0.05，***p<0.001。 A: Group-1, healthy control; B: Group-2, model control; C: Group-3 INT-747-20mg/kg, QD; D: Group-4, Pentoxifylline-100mg/kg, TID; E: group-5, compound 1-1mg/kg, BID; F: group-6, compound 1-3mg/kg, BID; G: group-7, compound 1-10mg/kg, BID; Figure 19: CCl 4 Induced mouse liver fibrosis model mouse liver pathological inflammation score. Data are indicated as mean±SEM, n=15. Compared with the model group, ***p<0.001, **p<0.01, *p<0.05; Figure 20: CCl 4 induced mouse liver fibrosis model mouse liver balloon-like variation value. The data are indicated by the mean±SEM, n=15, compared with the model group, ***p<0.001, **p<0.01, *p<0.05; Figure 21: CCl 4 induced mouse liver fibrosis model mice The score of liver watery degeneration. The data are indicated by the mean±SEM, n=15, compared with the model group, ***p<0.001, **p<0.01, *p<0.05; Figure 22: CCl 4 induced mouse liver fibrosis model mice Liver necrosis score. Data are indicated by mean±SEM, n=15, compared with the model group, ***p<0.001, **p<0.01, *p<0.05; Figure 23: CCl 4 induced mouse liver fibrosis model mice Comprehensive score for liver damage. The data are indicated by the mean±SEM, n=15, compared with the model group, ***p<0.001, **p<0.01, *p<0.05; Figure 24: CCl 4 induced mouse liver fibrosis model mice Liver Sirius Red stained picture, 50 times magnification: A: Group-1, healthy control; B: Group-2, model control; C: Group-3 INT-747-20mg/kg, QD; D: Group-4, self Ketoxifylline-100mg/kg, TID; E: group-5, compound 1-1mg/kg, BID; F: group-6, compound 1-3mg/kg, BID; G: group-7, compound 1-10mg /kg, BID; and Figure 25: CCl 4 induced mouse liver fibrosis model mouse liver fibrosis area. Data are indicated as mean±SEM, n=15, compared with the model group, *p<0.05, ***p<0.001.

下面通過實施例對本發明進行詳細描述，但並不意味著對本發明任何不利限制。 The following examples describe the present invention in detail, but they are not meant to impose any disadvantageous restriction on the present invention.

實施例1。 Example 1.

3，7-二甲基-1-(6，6，6-三氟-5-羥基-5-甲基己基)-1H-嘌呤-2，6(3H，7H)-二酮。 3,7-Dimethyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)-1 H -purine-2,6(3H,7H)-dione.

Figure 105135042-A0305-02-0027-237
Figure 105135042-A0305-02-0027-237

第一步。 first step.

3，7-二甲基-1-(6，6，6-三氟-5-甲基-5-(三甲基矽氧基)己基)-1H-嘌呤-2，6(3H，7H)-二酮。 3,7-Dimethyl-1-(6,6,6-trifluoro-5-methyl-5-(trimethylsiloxy)hexyl)-1 H -purine-2,6(3 H , 7 H )-Diketone.

將3，7-二甲基-1-(5-氧代己基)-1H-嘌呤-2，6(3H，7H)-二酮(200mg，0.719mmol)，氟化銫(10.9mg，0.0719mmol)溶於四氫呋喃(2mL)中，0℃下滴加三氟甲基三甲基矽烷(153mg，1.08mmol)。反應液在20℃下攪拌2小時，加入飽和食鹽水(50mL)淬滅反應，用乙酸乙酯萃取(100mL x 3)。有機相用飽和食鹽水洗滌(100mL x 3)，無水硫酸鈉乾燥後減壓濃縮。真空乾燥得到3，7-二甲基-1-(6，6，6-三氟-5-甲基-5-(三甲基矽氧基)己基)-1H-嘌呤-2，6(3H，7H)-二酮(200mg，白色固體)，產率：66%。MS-ESI計算值[M+H]+ 421，實測值421。 The 3,7-dimethyl-1-(5-oxohexyl)-1 H -purine-2,6(3 H , 7 H )-dione (200mg, 0.719mmol), cesium fluoride (10.9mg , 0.0719mmol) was dissolved in tetrahydrofuran (2mL), and trifluoromethyltrimethylsilane (153mg, 1.08mmol) was added dropwise at 0°C. The reaction solution was stirred at 20°C for 2 hours, and saturated brine (50 mL) was added to quench the reaction, and extracted with ethyl acetate (100 mL x 3). The organic phase was washed with saturated brine (100 mL x 3), dried over anhydrous sodium sulfate and concentrated under reduced pressure. Dry under vacuum to obtain 3,7-dimethyl-1-(6,6,6-trifluoro-5-methyl-5-(trimethylsiloxy)hexyl)-1 H -purine-2,6( 3 H , 7 H )-dione (200 mg, white solid), yield: 66%. MS-ESI calculated value [M+H] + 421, measured value 421.

第二步。 The second step.

3，7-二甲基-1-(6，6，6-三氟-5-羥基-5-甲基己基)-1H-嘌呤-2，6(3H，7H)-二酮。 3,7-dimethyl-1- (6,6,6-trifluoro-5-hydroxy-5-methylhexyl) -1 H - purine -2,6 (3 H, 7 H) - dione.

將3，7-二甲基-1-(6，6，6-三氟-5-甲基-5-(三甲基矽氧基)己基)-1H-嘌呤-2，6(3H，7H)-二酮(200mg，0.476mmol)溶於四氫呋喃(2mL)，0℃下滴加1M鹽酸(0.5mL)後於20℃攪拌1小時。混合物冷却到0℃，加入碳酸氫鈉溶液(30mL)淬滅反應。混合物用乙酸乙酯(100mL x 3)萃取。有機相用飽和食鹽水(100mL x 3)洗滌，無水硫酸鈉乾燥後減壓濃縮。用製備高效液相色譜分離純化得到3，7-二甲基-1-(6，6，6-三氟-5-羥基-5-甲基己基)-1H-嘌呤-2，6(3H，7H)-二酮(50.0mg，白色固體)，產率：30%。1H NMR：(400MHz，Methonal-d 4 )δ7.85(s，1H)，4.02-3.98(m，2H)，3.96(s，3H)，3.52(s，3H)，1.69-1.64(m，4H)，1.52-1.48(m，2H)，1.28(s，3H)。MS-ESI計算值[M+H]+ 349，實測值349。 3,7-Dimethyl-1-(6,6,6-trifluoro-5-methyl-5-(trimethylsiloxy)hexyl)-1 H -purine-2,6(3H, 7H)-dione (200mg, 0.476mmol) was dissolved in tetrahydrofuran (2mL), 1M hydrochloric acid (0.5mL) was added dropwise at 0°C, and the mixture was stirred at 20°C for 1 hour. The mixture was cooled to 0°C, and sodium bicarbonate solution (30 mL) was added to quench the reaction. The mixture was extracted with ethyl acetate (100 mL x 3). The organic phase was washed with saturated brine (100 mL x 3), dried over anhydrous sodium sulfate and concentrated under reduced pressure. Separated and purified by preparative high performance liquid chromatography to obtain 3,7-dimethyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)-1 H -purine-2,6(3 H , 7 H )-diketone (50.0 mg, white solid), yield: 30%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.85 (s, 1H), 4.02-3.98 (m, 2H), 3.96 (s, 3H), 3.52 (s, 3H), 1.69-1.64 (m, 4H), 1.52-1.48 (m, 2H), 1.28 (s, 3H). MS-ESI calculated value [M+H] + 349, measured value 349.

實施例2。 Example 2.

1-(5-羥基-5-甲基庚基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1- (5-hydroxy-5-methyl-heptyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

Figure 105135042-A0305-02-0028-46
Figure 105135042-A0305-02-0028-46

第一步。 first step.

乙基5-(3，7-二甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)戊酸乙酯。 Ethyl 5-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)pentanoic acid ethyl ester.

將3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(5.00g，28.0mmol)，溴戊酸乙酯(7.51g，33.4mmol)，碳酸鉀(7.73g，56.0mmol)和碘化鉀(500mg，2.80mmol)溶解於N，N-二甲基甲醯胺(62mL)中。反應液加熱到110℃，攪拌兩小時。把反應倒入水中，用乙酸乙酯萃取(20mL x 3)。合併有機相，用無水硫酸鈉乾燥，過濾，濾液濃縮得乙基5-(3，7-二甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)戊酸乙酯(5.00g，黃色固體)，產率：50%。1H NMR：(400MHz，CDCl3)δ7.51(s，1H)，4.14-4.09(m，2H)，4.04-4.01(m，2H)，3.97(s，3H)，3.57(s，3H)，2.37-2.33(m，2H)，1.72-1.69(m，4H)，1.25(t，J=7.2Hz，3H)。 The 3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (5.00g, 28.0mmol), bromo-pentanoate (7.51g, 33.4mmol), potassium carbonate (7.73 g, 56.0 mmol) and potassium iodide (500 mg, 2.80 mmol) were dissolved in N , N -dimethylformamide (62 mL). The reaction solution was heated to 110°C and stirred for two hours. The reaction was poured into water and extracted with ethyl acetate (20 mL x 3). The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain ethyl 5-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H- Purin-1-yl) ethyl valerate (5.00 g, yellow solid), yield: 50%. 1 H NMR: (400MHz, CDCl 3 ) δ7.51 (s, 1H), 4.14 to 4.09 (m, 2H), 4.04-4.01 (m, 2H), 3.97 (s, 3H), 3.57 (s, 3H) , 2.37-2.33 (m, 2H), 1.72-1.69 (m, 4H), 1.25 (t, J = 7.2 Hz, 3H).

第二步。 The second step.

1-(5-乙基-5-羥基庚基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1- (5-ethyl-5-hydroxy-heptyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

將乙基5-(3，7-二甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)戊酸乙酯(0.500g，1.62mmol)溶解於無水四氫呋喃(5mL)中，氮氣保護，-78℃條件下慢慢滴入乙基溴化鎂(3M乙醚溶液，3.42mL，9.72mmol)。反應液在-78℃攪拌0.5小時，慢慢升至然後0℃，反應0.5小時。將反應液倒入水中，用乙酸乙酯萃取(30mL x 3)。合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮得1-(5-乙基-5-羥基庚基-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(0.300g，無色油狀物)，產率：57%。1H NMR：(400MHz，CDCl3)δ7.50(s，1H)，4.05-4.01(m，2H)，3.99(s，3H)，3.57(s，3H)，1.70-1.37(m，10H)，0.86(t，J=7.6Hz，6H)。MS-ESI計算值[M+H]+ 323，實測值323。 Ethyl 5-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)pentanoic acid ethyl ester (0.500g, 1.62mmol) was dissolved in anhydrous tetrahydrofuran (5mL), and under nitrogen protection, ethylmagnesium bromide (3M ether solution, 3.42mL, 9.72mmol) was slowly added dropwise at -78°C. The reaction solution was stirred at -78°C for 0.5 hours, slowly raised to 0°C, and reacted for 0.5 hours. The reaction solution was poured into water, and extracted with ethyl acetate (30 mL x 3). The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 1-(5-ethyl-5-hydroxyheptyl-3,7-dimethyl-1 H -purine-2,6(3 H , 7 H )-dione (0.300 g, colorless oil), yield: 57%. 1 H NMR: (400MHz, CDCl 3 ) δ 7.50 (s, 1H), 4.05-4.01 (m, 2H) , 3.99 (s, 3H), 3.57 (s, 3H), 1.70-1.37 (m, 10H), 0.86 (t, J = 7.6 Hz, 6H). MS-ESI calculated value [M+H] + 323, measured The value is 323.

實施例3。 Example 3.

1-(4-(1-羥基環丙基)丁基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1- (4- (1-hydroxy-cyclopropyl) butyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

Figure 105135042-A0305-02-0030-50
Figure 105135042-A0305-02-0030-50

氮氣保護下，-35℃將乙基溴化鎂(3M乙醚溶劑，1.1mL，3.24mmol)加入到乙基5-(3，7-二甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)戊酸乙酯(500mg，1.62mmol)和鈦酸四異丙酯(461mg，1.62mmol)的四氫呋喃(10mL)溶液中。反應液緩慢加熱至25℃，攪拌2小時。加入水(10mL)淬滅反應，過濾除去不溶物，濾液用乙酸乙酯萃取(20mL x 3)。合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮用高效液相色譜法分離純化得到1-(4-(1-羥基環丙基)丁基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(90.0mg，白色固體)，產率：19%。1H NMR：(400MHz，Methonal-d 4)δ7.86(s，1H)，4.03-3.90(m，5H)，3.51(s，3H)，1.72-1.53(m，6H)，0.68-0.59(m，2H)，0.46-0.38(m，2H)。MS-ESI計算值[M+H]+ 293，實測值293。 Under the protection of nitrogen, add ethyl magnesium bromide (3M ether solvent, 1.1 mL, 3.24 mmol) to ethyl 5-(3,7-dimethyl-2,6-dioxo-2,3 at -35°C). , 6,7-Tetrahydro- 1H -purin-1-yl)ethyl valerate (500mg, 1.62mmol) and tetraisopropyl titanate (461mg, 1.62mmol) in tetrahydrofuran (10mL). The reaction solution was slowly heated to 25°C and stirred for 2 hours. Water (10 mL) was added to quench the reaction, the insoluble matter was removed by filtration, and the filtrate was extracted with ethyl acetate (20 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated and purified by high performance liquid chromatography to obtain 1-(4-(1-hydroxycyclopropyl)butyl)-3,7-dimethyl- 1 H - purine -2,6 (3 H, 7 H) - dione (90.0 mg, white solid), yield: 19%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.86 (s, 1H), 4.03-3.90 (m, 5H), 3.51 (s, 3H), 1.72-1.53 (m, 6H), 0.68-0.59 ( m, 2H), 0.46-0.38 (m, 2H). MS-ESI calculated value [M+H] + 293, measured value 293.

實施例4。 Example 4.

3，7-二甲基-1-((1-(1，1，1-三氟-2-羥基丙烷-2-基)環丙基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3,7-Dimethyl-1-((1-(1,1,1-trifluoro-2-hydroxypropan-2-yl)cyclopropyl)methyl)-1 H -purine-2,6- (3 H , 7 H )-dione.

Figure 105135042-A0305-02-0030-48
Figure 105135042-A0305-02-0030-48
Figure 105135042-A0305-02-0031-53
Figure 105135042-A0305-02-0031-53

第一步。 first step.

乙基1-乙醯基環丙烷。 Ethyl 1-acetylcyclopropane.

將乙基-3-氧代丁酸(10.0g，76.8mmol)和1，2-二溴乙烷(21.7g，115mmol)溶於二甲基亞碸(300mL)中，在氮氣保護下，再分批加入碳酸鉀(42.5g，307mmol)。將反應液置於25℃攪拌24小時。加入水(500mL)，反應液用乙酸乙酯(300mL x 3)萃取，合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法純化(10：1石油醚/乙酸乙酯，Rf=0.4)得到乙基1-乙醯基環丙烷(6.00g，白色油狀物)，產率：50%。1H NMR：(400MHz，Methonal-d 4 )δ4.25-4.20(m，2H)，2.44(s，3H)，1.47-1.42(m，4H)，1.32-1.28(m，3H)。 Ethyl-3-oxobutyric acid (10.0g, 76.8mmol) and 1,2-dibromoethane (21.7g, 115mmol) were dissolved in dimethyl sulfoxide (300mL), and then under nitrogen protection. Potassium carbonate (42.5 g, 307 mmol) was added in portions. The reaction solution was placed at 25°C and stirred for 24 hours. Water (500mL) was added, the reaction solution was extracted with ethyl acetate (300mL x 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified by silica gel column chromatography (10:1 petroleum ether/acetic acid) Ethyl ester, Rf=0.4) to obtain ethyl 1-acetylcyclopropane (6.00 g, white oil), yield: 50%. 1 H NMR: (400 MHz, Methonal- d 4 ) δ 4.25-4.20 (m, 2H), 2.44 (s, 3H), 1.47-1.42 (m, 4H), 1.32-1.28 (m, 3H).

第二步。 The second step.

1-(1，1，1-三氟-2-羥基丙烷-2-基)環丙烷羧酸。 1-(1,1,1-trifluoro-2-hydroxypropan-2-yl)cyclopropane carboxylic acid.

將乙基1-乙醯基環丙烷(2.00g，12.8mmol)，氟化銫(195mg，1.28mmol)溶於四氫呋喃(30mL)中，然後0℃加入三氟甲基三甲基矽烷(3.64g，25.6mmol)。反應液在20℃氮氣保護下反應6小時。然後加入4N稀鹽酸(7mL)。混合物在室溫氮氣保護下反應6小時。加入碳酸氫鈉飽和溶液(30mL)淬滅反應，用乙酸乙酯萃取(100mL x 3)，有機相用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法純化(10：1石油醚/乙酸乙酯，Rf=0.4)得到1-(1，1，1-三氟-2-羥基丙烷-2-基)環丙烷羧酸 (1.70g，白色油狀物)，產率：59%。1H NMR：(400MHz，Methonal-d 4 )δ4.14-4.10(m，2H)，1.64(s，3H)，1.29-1.24(m，3H)，1.23-1.22(m，2H)，0.92-0.90(m，2H)。 Dissolve ethyl 1-acetylcyclopropane (2.00g, 12.8mmol) and cesium fluoride (195mg, 1.28mmol) in tetrahydrofuran (30mL), then add trifluoromethyltrimethylsilane (3.64g) at 0°C , 25.6mmol). The reaction solution was reacted at 20°C under nitrogen protection for 6 hours. Then 4N dilute hydrochloric acid (7 mL) was added. The mixture was reacted for 6 hours under nitrogen protection at room temperature. The reaction was quenched by adding saturated sodium bicarbonate solution (30mL), extracted with ethyl acetate (100mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified by silica gel column chromatography (10:1 petroleum Ether/ethyl acetate, Rf=0.4) to obtain 1-(1,1,1-trifluoro-2-hydroxypropan-2-yl)cyclopropanecarboxylic acid (1.70g, white oil), yield: 59 %. 1 H NMR: (400MHz, Methonal- d 4 ) δ 4.14-4.10 (m, 2H), 1.64 (s, 3H), 1.29-1.24 (m, 3H), 1.23-1.22 (m, 2H), 0.92- 0.90 (m, 2H).

第三步。 third step.

1，1，1-三氟-2-(1-(羥甲基)環丙基)丙-2-醇。 1,1,1-Trifluoro-2-(1-(hydroxymethyl)cyclopropyl)propan-2-ol.

將1-(1，1，1-三氟-2-羥基丙烷-2-基)環丙烷羧酸(400mg，1.77mmol)溶於無水四氫呋喃(10mL)中，0℃下加入四氫鋁鋰(81.0mg，2.12mmol)。反應液升溫至25℃，攪拌1小時。加水(10mL)淬滅，用乙酸乙酯(50mL x 3)萃取，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法純化(1：1石油醚/乙酸乙酯，Rf=0.2)，得到1，1，1-三氟-2-(1-(羥甲基)環丙基)丙-2-醇(200mg，黃色油狀物)，產率：61%。1H NMR：(400MHz，DMSO-d 6 )δ5.64(s，1H)，4.63-4.60(m，1H)，3.64-3.60(m，1H)，3.23-3.17(m，1H)，1.36(s，3H)，0.83-0.91(m，1H)，0.56-0.55(m，1H)，0.39-0.35(m，2H)。 Dissolve 1-(1,1,1-trifluoro-2-hydroxypropan-2-yl)cyclopropanecarboxylic acid (400mg, 1.77mmol) in anhydrous tetrahydrofuran (10mL), add lithium tetrahydroaluminum ( 81.0mg, 2.12mmol). The reaction solution was heated to 25°C and stirred for 1 hour. Quench with water (10mL), extract with ethyl acetate (50mL x 3), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify with silica gel column chromatography (1:1 petroleum ether/ethyl acetate, Rf=0.2 ) To obtain 1,1,1-trifluoro-2-(1-(hydroxymethyl)cyclopropyl)propan-2-ol (200 mg, yellow oil), yield: 61%. 1 H NMR: (400MHz, DMSO- d 6 ) δ 5.64 (s, 1H), 4.63-4.60 (m, 1H), 3.64-3.60 (m, 1H), 3.23-3.17 (m, 1H), 1.36 ( s, 3H), 0.83-0.91 (m, 1H), 0.56-0.55 (m, 1H), 0.39-0.35 (m, 2H).

第四步。 the fourth step.

(1-(1，1，1-三氟-2-羥基丙烷-2-基)環丙基)甲基甲磺酸酯 (1-(1,1,1-Trifluoro-2-hydroxypropan-2-yl)cyclopropyl)methanesulfonate

將1，1，1-三氟-2-(1-(羥甲基)環丙基)丙-2-醇(100mg，0.543mmol)溶解在二氯甲烷(5mL)中，在0℃下加入三乙胺(110mg，1.08mmol)和甲烷磺醯氯(62.2mg，0.543mmol)。反應液在0℃下反應2小時。加入碳酸氫鈉飽和水溶液(10mL)淬滅，用二氯甲烷(10mL x 3)萃取，合併有機相，用飽和氯化鈉溶液(10mL x 3)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到(1-(1，1，1-三氟-2-羥基丙烷-2-基)環丙基)甲基甲磺酸酯(80.0mg，黃色油狀物)，產率：56%。 Dissolve 1,1,1-trifluoro-2-(1-(hydroxymethyl)cyclopropyl)propan-2-ol (100mg, 0.543mmol) in dichloromethane (5mL) and add at 0°C Triethylamine (110 mg, 1.08 mmol) and methanesulfonyl chloride (62.2 mg, 0.543 mmol). The reaction solution was reacted at 0°C for 2 hours. It was quenched by adding saturated aqueous sodium bicarbonate (10 mL), extracted with dichloromethane (10 mL x 3), combined the organic phases, washed with saturated sodium chloride solution (10 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was reduced in pressure Concentrate to obtain (1-(1,1,1-trifluoro-2-hydroxypropan-2-yl)cyclopropyl)methanesulfonate (80.0mg, yellow oil), yield: 56% .

第五步。 the fifth step.

3，7-二甲基-1-((1-(1，1，1-三氟-2-羥基丙烷-2-基)環丙基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3,7-Dimethyl-1-((1-(1,1,1-trifluoro-2-hydroxypropan-2-yl)cyclopropyl)methyl)-1 H -purine-2,6- (3 H , 7 H )-dione.

(1-(1，1，1-三氟-2-羥基丙烷-2-基)環丙基)甲基甲磺酸酯(80.0mg，0.305mmol)，3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(54.9mg，0.305mmol)，碘化鉀(5.10mg，0.0305mmol)和碳酸鉀(126mg，0.915mmol)溶於無水N，N-二甲基甲醯胺(5mL)中。反應液加熱至120℃，反應2小時。反應液冷却至20℃，過濾，用製備高效液相色譜純化，得到3，7-二甲基-1-((1-(1，1，1-三氟-2-羥基丙烷-2-基)環丙基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮(40.0mg，白色固體)，產率：38%。1H NMR：(400MHz，Methonal-d 4 )δ7.88(s，1H)，4.45(d，J=6.8Hz，1H)，4.24(d，J=6.8Hz，1H)，3.97(s，3H)，3.53(s，3H)，1.53(s，3H)，0.92-0.88(m，1H)，0.64-0.63(m，1H)，0.41-0.38(m，1H)，0.15-0.12(m，1H)。 (1-(1,1,1-Trifluoro-2-hydroxypropan-2-yl)cyclopropyl)methanesulfonate (80.0mg, 0.305mmol), 3,7-dimethyl-1 H - purine -2,6- (3 H, 7 H) - dione (54.9mg, 0.305mmol), potassium iodide (5.10mg, 0.0305mmol) and potassium carbonate (126mg, 0.915mmol) was dissolved in anhydrous N, N - two Methylformamide (5mL). The reaction solution was heated to 120°C and reacted for 2 hours. The reaction solution was cooled to 20°C, filtered, and purified by preparative high performance liquid chromatography to obtain 3,7-dimethyl-1-((1-(1,1,1-trifluoro-2-hydroxypropan-2-yl ) cyclopropyl) methyl) -1 H - purine -2,6- (3 H, 7 H) - dione (40.0 mg, white solid), yield: 38%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.88 (s, 1H), 4.45 (d, J = 6.8 Hz, 1H), 4.24 (d, J = 6.8 Hz, 1H), 3.97 (s, 3H) ), 3.53(s, 3H), 1.53(s, 3H), 0.92-0.88(m, 1H), 0.64-0.63(m, 1H), 0.41-0.38(m, 1H), 0.15-0.12(m, 1H) ).

MS-ESI計算值[M+H]+ 347，實測值347。 MS-ESI calculated value [M+H] + 347, measured value 347.

實施例5。 Example 5.

1-((3-羥基-3-(三氟甲基)環丁基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1-((3-Hydroxy-3-(trifluoromethyl)cyclobutyl)methyl)-3,7-dimethyl-1 H -purine-2,6(3 H ,7 H )-dione .

Figure 105135042-A0305-02-0033-54
Figure 105135042-A0305-02-0033-54
Figure 105135042-A0305-02-0034-55
Figure 105135042-A0305-02-0034-55

第一步。 first step.

3-氧代環丁烷羧酸甲酯。 Methyl 3-oxocyclobutanecarboxylate.

將3-氧代環丁烷羧酸(25.0g，0.220mmol)，甲醇(14mL)及N，N-二甲基-4-氨基吡啶(3.00g，353mmol)溶於二氯甲烷(500mL)中，25℃攪拌，慢慢滴加1-(3-二甲氨基丙基)-3-乙基碳二亞胺鹽酸鹽(64.0g，340mmol)，攪拌過夜。反應液依次用鹽酸水溶液(1.5N，72mL)，水(150mL x 2)和飽和食鹽水(75mL x 2)洗滌。有機相用無水硫酸鈉乾燥，減壓濃縮得到產品3-氧代環丁烷羧酸甲酯(25g，黃色液體)，產率：89%。 Dissolve 3-oxocyclobutanecarboxylic acid (25.0g, 0.220mmol), methanol (14mL) and N , N -dimethyl-4-aminopyridine (3.00g, 353mmol) in dichloromethane (500mL) , Stir at 25°C, slowly add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (64.0 g, 340 mmol) dropwise, and stir overnight. The reaction solution was washed sequentially with aqueous hydrochloric acid (1.5N, 72 mL), water (150 mL x 2) and saturated brine (75 mL x 2). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the product methyl 3-oxocyclobutanecarboxylate (25 g, yellow liquid), yield: 89%.

第二步。 The second step.

5，8-二氧雜螺[3，4]辛烷-2-羧酸甲酯。 5,8-Dioxaspiro[3,4]octane-2-carboxylic acid methyl ester.

將甲基-3-氧代環丁烷羧酸(25.0g，195mmol)，乙二醇(35.0g，564mmol)和對甲苯磺酸(3.50g，20.0mmol)溶於甲苯(250mL)中，加分水器後加熱回流過夜。反應液冷却至25℃，依次用水(300mL x 2)、飽和碳酸氫鈉(500mL x 2)洗滌。有機相用無水硫酸鎂乾燥，過濾，濾液 減壓濃縮得到產物甲基5，8-二氧雜螺[3，4]辛烷-2-羧酸甲酯(22.5g，黃色液體)，產率：90%。 Methyl-3-oxocyclobutanecarboxylic acid (25.0g, 195mmol), ethylene glycol (35.0g, 564mmol) and p-toluenesulfonic acid (3.50g, 20.0mmol) were dissolved in toluene (250mL) and added After the water separator, heat to reflux overnight. The reaction solution was cooled to 25°C, and washed with water (300 mL x 2) and saturated sodium bicarbonate (500 mL x 2) in sequence. The organic phase was dried with anhydrous magnesium sulfate, filtered, and the filtrate Concentration under reduced pressure gave the product methyl 5,8-dioxaspiro[3,4]octane-2-carboxylate (22.5g, yellow liquid), yield: 90%.

第三步。 third step.

5，8-二氧雜螺[3，4]辛烷-2-甲醇。 5,8-Dioxaspiro[3,4]octane-2-methanol.

在氮氣保護，0℃時下將四氫鋁鋰(5.20g，136mmol)緩慢溶於四氫呋喃(240mL)中，然後滴加溶於四氫呋喃(60mL)中的5，8-二氧雜螺[3，4]辛烷-2-羧酸甲酯(19.5g，113mmol)。反應緩慢升至25℃，攪拌3.5小時。反應液冷却至0℃，依次緩慢加入水(5.20g，289mmol)，15%氫氧化鈉(5.20g，19.5mmol)及水(15.6g，867mmol)。過濾，濾餅用四氫呋喃(10mL x 3)洗滌，濾液減壓濃縮用矽膠柱色譜法純化(1：1石油醚/乙酸乙酯，Rf=0.4)得到產物5，8-二氧雜螺[3，4]辛烷-2-甲醇(10.0g，黃色液體)，產率：62%。1H NMR：(400MHz，CDCl3)δ3.90-3.87(m，4H)，3.67(d，J=6.4Hz，2H)，2.45-2.40(m，2H)，2.38-2.26(m，1H)，2.13-2.08(m，2H)。 Under the protection of nitrogen, lithium tetrahydroaluminum (5.20g, 136mmol) was slowly dissolved in tetrahydrofuran (240mL) at 0℃, and then 5,8-dioxaspiro [3, 4] Methyl octane-2-carboxylate (19.5 g, 113 mmol). The reaction was slowly raised to 25°C and stirred for 3.5 hours. The reaction solution was cooled to 0°C, and water (5.20 g, 289 mmol), 15% sodium hydroxide (5.20 g, 19.5 mmol) and water (15.6 g, 867 mmol) were slowly added in sequence. Filtration, the filter cake was washed with tetrahydrofuran (10mL x 3), the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (1:1 petroleum ether/ethyl acetate, Rf=0.4) to obtain the product 5,8-dioxaspiro [3 , 4] Octane-2-methanol (10.0 g, yellow liquid), yield: 62%. 1 H NMR: (400MHz, CDCl 3 ) δ 3.90-3.87 (m, 4H), 3.67 (d, J = 6.4 Hz, 2H), 2.45-2.40 (m, 2H), 2.38-2.26 (m, 1H) , 2.13-2.08 (m, 2H).

第四步。 the fourth step.

5，8-二氧雜螺[3，4]辛烷-2-基甲基甲磺酸酯。 5,8-Dioxaspiro[3,4]octane-2-yl methanesulfonate.

將5，8-二氧雜螺[3，4]辛烷-2-甲醇(500mg，53.1mmol)及三乙胺(896mg，6.90mmol)溶於二氯甲烷(23mL)，在0℃緩慢加入甲烷磺醯氯(1.40g，12.6mmol)。反應液升至在25℃，攪拌過夜。加入水(50mL)淬滅反應，用乙酸乙酯萃取(50mL x 3)。合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到產物5，8-二氧雜螺[3，4]辛烷-2-基甲基甲磺酸酯(2.30g，黃色液體)。 Dissolve 5,8-dioxaspiro[3,4]octane-2-methanol (500mg, 53.1mmol) and triethylamine (896mg, 6.90mmol) in dichloromethane (23mL), add slowly at 0°C Methanesulfonyl chloride (1.40 g, 12.6 mmol). The reaction solution was raised to 25°C and stirred overnight. The reaction was quenched by adding water (50 mL) and extracted with ethyl acetate (50 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the product 5,8-dioxaspiro[3,4]octane-2-ylmethyl methanesulfonate (2.30g, yellow liquid) .

MS-ESI計算值[M+H]+ 223，實測值223。 MS-ESI calculated value [M+H] + 223, measured value 223.

第五步。 the fifth step.

(1-(5，8-二氧雜螺[3，4]辛烷-2-基甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮 (1-(5,8-Dioxaspiro[3,4]octan-2-ylmethyl)-3,7-dimethyl-1 H -purine-2,6(3 H ,7 H ) -Diketone

將5，8-二氧雜螺[3，4]辛烷-2-基甲基甲磺酸酯(1.00g，4.50mmol)，3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(810mg，4.50mmol)，碳酸鉀(1.20g，13.5mmol)和碘化鉀(75.0mg，0.45mmol)溶於N，N-二甲基甲醯胺(20mL)中。反應加熱至130℃，攪拌3.5小時。反應液過濾，濾液減壓濃縮，得到1-(5，8-二氧雜螺[3，4]辛烷-2-基甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(1.50g，棕色液體)，產率：93%。MS-ESI計算值[M+H]+ 307，實測值307。 5,8-Dioxaspiro[3,4]octane-2-ylmethyl methanesulfonate (1.00g, 4.50mmol), 3,7-dimethyl- 1H -purine-2,6 (3 H , 7 H )-dione (810mg, 4.50mmol), potassium carbonate (1.20g, 13.5mmol) and potassium iodide (75.0mg, 0.45mmol) dissolved in N , N -dimethylformamide (20mL) in. The reaction was heated to 130°C and stirred for 3.5 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain 1-(5,8-dioxaspiro[3,4]octan-2-ylmethyl)-3,7-dimethyl-1 H -purine-2 , 6 (3 H, 7 H ) - dione (1.50 g of, brown liquid), yield: 93%. MS-ESI calculated value [M+H] + 307, measured value 307.

第六步。 The sixth step.

3，7-二甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮。 3,7-dimethyl-1 - ((4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione.

將1-(5，8-二氧雜螺[3，4]辛烷-2-基甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(1.50g，5.00mmol)溶於丙酮(18mL)中，加入鹽酸水溶液(4N，3mL)。反應加熱至30℃，攪拌過夜。加入水稀釋，用飽和碳酸氫鈉水溶液(20mL)調節pH至中性，用乙酸乙酯萃取(150mL x 3)。合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到的產品用矽膠柱色譜法純化(1：1石油醚/乙酸乙酯，Rf=0.2)得到產物3，7-二甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮(180mg，白色固體)，產率：14%。 1- (5,8-dioxaspiro [3,4] octane-2-yl-methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) -Dione (1.50 g, 5.00 mmol) was dissolved in acetone (18 mL), and aqueous hydrochloric acid (4N, 3 mL) was added. The reaction was heated to 30°C and stirred overnight. Add water to dilute, adjust the pH to neutral with saturated sodium bicarbonate aqueous solution (20 mL), and extract with ethyl acetate (150 mL x 3). The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product obtained was purified by silica gel column chromatography (1:1 petroleum ether/ethyl acetate, Rf=0.2) to obtain the product 3,7-dimethyl -1 - ((4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (180 mg of, white solid), yield: 14%.

1H NMR：(400MHz，CDCl3)δ7.49(s，1 H)，4.25(d，J=7.6Hz，2 H)，3.95(s，3 H)，3.55(s，3 H)，3.13-2.96(m，4 H)，2.95-2.84(m，1 H)。MS-ESI計算值[M+H]+ 263，實測值263。 1 H NMR: (400MHz, CDCl 3 )δ7.49 (s, 1 H), 4.25 (d, J = 7.6 Hz, 2 H), 3.95 (s, 3 H), 3.55 (s, 3 H), 3.13 -2.96 (m, 4 H), 2.95 to 2.84 (m, 1 H). MS-ESI calculated value [M+H] + 263, measured value 263.

第七步。 The seventh step.

1-((3-羥基-3-(三氟甲基)環戊基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1-((3-Hydroxy-3-(trifluoromethyl)cyclopentyl)methyl)-3,7-dimethyl-1 H -purine-2,6(3 H ,7 H )-dione .

將3，7-二甲基-1-((3-氧代環戊基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮(100mg，0.382mmol)和氟化銫(11.5mg，0.0763mmol)溶於無水四氫呋喃(3mL)中，氮氣保護下加入三氟甲基三甲基矽烷(95.0mg，0.640mmol)。反應液緩慢加熱至30℃下，攪拌12小時。然後向反應中加入鹽酸水溶液(1N，5mL)繼續攪拌0.5小時。向反應液中加入水(50mL)稀釋，用飽和碳酸氫鈉水溶液(10mL)調pH值至7，減壓濃縮，用製備高效液相色譜純化，得到1-((3-羥基-3-(三氟甲基)環戊基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(80.0mg，白色固體)，產率：64%。1H NMR：(400MHz，Mehonal-d 4 )δ8.54(s，1H)，4.13-4.07(m，5H)，3.56(s，3H)，2.58-2.48(m，3H)，2.14-2.10(m，2H)。MS-ESI計算值[M+H]+ 333，實測值333。 3,7-dimethyl-1 - ((3-oxo-cyclopentyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (100mg, 0.382mmol) and Cesium fluoride (11.5mg, 0.0763mmol) was dissolved in anhydrous tetrahydrofuran (3mL), and trifluoromethyltrimethylsilane (95.0mg, 0.640mmol) was added under nitrogen protection. The reaction solution was slowly heated to 30°C and stirred for 12 hours. Then an aqueous hydrochloric acid solution (1N, 5 mL) was added to the reaction and stirring was continued for 0.5 hour. The reaction solution was diluted with water (50 mL), adjusted to pH 7 with saturated sodium bicarbonate aqueous solution (10 mL), concentrated under reduced pressure, and purified by preparative high performance liquid chromatography to obtain 1-((3-hydroxy-3-( trifluoromethyl) cyclopentyl) methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (80.0 mg, white solid), yield: 64%. 1H NMR: (400MHz, Mehonal- d 4 ) δ 8.54 (s, 1H), 4.13-4.07 (m, 5H), 3.56 (s, 3H), 2.58-2.48 (m, 3H), 2.14-2.10 (m , 2H). MS-ESI calculated value [M+H] + 333, measured value 333.

實施例6。 Example 6.

1-((3，7-二甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)-3-羥基環丁腈。 1-((3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -purin-1-yl)methyl)-3-hydroxycyclobutyronitrile .

Figure 105135042-A0305-02-0037-56
Figure 105135042-A0305-02-0037-56

第一步。 first step.

(((1，3-二溴丙-2-基)氧基)甲基)苯。 (((1,3-Dibromoprop-2-yl)oxy)methyl)benzene.

將2-(溴甲基)環氧乙烷(8.40g，61.3mmol)室溫下加入到溶有氯化亞銅(6.87g，51.1mmol)的苄溴(8.74g，51.1mmol)中。反應150℃攪拌11小時。反應液冷却至室溫，緩慢加入水(100mL)並用乙酸乙酯(100mL x 3)萃取。合併有機相，並用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到的產品用矽膠柱色譜法純化(石油醚，Rf=0.6)得到產物(((1，3-二溴丙-2-基)氧基)甲基)苯(8.60g，黃色油狀)，產率：44%。1H NMR：(400MHz，CDCl3)δ7.39-7.31(m，5H)，4.67(s，2H)，3.82-3.78(m，1H)，3.58(d，J=5.2Hz，4H)。 2-(Bromomethyl)oxirane (8.40g, 61.3mmol) was added to benzyl bromide (8.74g, 51.1mmol) dissolved in cuprous chloride (6.87g, 51.1mmol) at room temperature. The reaction was stirred at 150°C for 11 hours. The reaction solution was cooled to room temperature, water (100 mL) was slowly added and extracted with ethyl acetate (100 mL x 3). The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product obtained was purified by silica gel column chromatography (petroleum ether, Rf=0.6) to obtain the product (((1,3-dibromoprop-2-yl) )Oxy)methyl)benzene (8.60g, yellow oil), yield: 44%. 1 H NMR: (400 MHz, CDCl 3 ) δ 7.39-7.31 (m, 5H), 4.67 (s, 2H), 3.82-3.78 (m, 1H), 3.58 (d, J = 5.2 Hz, 4H).

第二步。 The second step.

乙基3-(苄氧基)-1-氰基環丁烷羧酸乙酯。 Ethyl 3-(benzyloxy)-1-cyanocyclobutanecarboxylate.

將氰乙酸乙酯(2.76g，24.3mmol)室溫下緩慢加入到溶有(((1，3-二溴丙-2-基)氧基)甲基)苯(7.00g，18.2mmol)及碳酸鉀(10.0g，72.7mmol)的N，N-二甲基甲醯胺(35mL)中。反應90℃攪拌4小時。冷却至室溫，過濾，固體用乙酸乙酯(20mL)洗滌。得到的有機相用飽和氯化銨水溶液(20mL x 3)洗滌。有機相用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到的產品用矽膠柱色譜法純化(30：1石油醚/乙酸乙酯，Rf=0.4)得到產物乙基3-(苄氧基)-1-氰基環丁烷羧酸乙酯(3.80g，無色油狀)，產率：81%。1H NMR：(400MHz，Methonal-d 4)δ7.40-7.28(m，5H)，4.48-4.44(m，2H)，4.37-4.31(m，1H)，4.30-4.24(m，2H)，2.97-2.80(m，2H)，2.73-2.65(m，2H)，1.37-1.30(m，3H)。 Ethyl cyanoacetate (2.76g, 24.3mmol) was slowly added to the dissolved (((1,3-dibromoprop-2-yl)oxy)methyl)benzene (7.00g, 18.2mmol) and Potassium carbonate (10.0 g, 72.7 mmol) in N , N -dimethylformamide (35 mL). The reaction was stirred at 90°C for 4 hours. Cool to room temperature, filter, and wash the solid with ethyl acetate (20 mL). The resulting organic phase was washed with saturated aqueous ammonium chloride solution (20 mL x 3). The organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product obtained was purified by silica gel column chromatography (30:1 petroleum ether/ethyl acetate, Rf=0.4) to obtain the product ethyl 3-(benzyloxy) Ethyl-1-cyanocyclobutanecarboxylate (3.80 g, colorless oil), yield: 81%. 1 H NMR: (400MHz, Methonal- d 4 ) δ7.40-7.28 (m, 5H), 4.48-4.44 (m, 2H), 4.37-4.31 (m, 1H), 4.30-4.24 (m, 2H), 2.97-2.80 (m, 2H), 2.73-2.65 (m, 2H), 1.37-1.30 (m, 3H).

第三步。 third step.

3-(苄氧基)-1-(羥甲基)環丁腈。 3-(Benzyloxy)-1-(hydroxymethyl)cyclobutyronitrile.

將硼氫化鈉(1.39g，36.6mmol)溶於四氫呋喃和水(20mL：2mL)中，並在0℃下，20分鐘內緩慢滴加乙基3-(苄氧基)-1-氰基環丁烷羧酸乙酯(3.80g，14.6mmol)的四氫呋喃(22mL)溶液。反應室溫攪拌2小時。加入乙酸乙酯(50mL)稀釋，有機相分別用水(30mL)及飽和食鹽水(30mL)洗滌，並用無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到粗產品3-(苄氧基)-1-(羥甲基)環丁腈(3.70g，無色油狀)。1H NMR：(400MHz，DMSO-d 6)δ7.38-7.25(m，5H)，5.57-5.52(m，1H)，4.39-4.36(m，2H)，4.13-4.04(m，1H)，3.57-3.51(m，2H)，2.58-2.51(m，1H)，2.49-2.45(m，1H)，2.31-2.09(m，2H)。 Sodium borohydride (1.39g, 36.6mmol) was dissolved in tetrahydrofuran and water (20mL: 2mL), and ethyl 3-(benzyloxy)-1-cyano ring was slowly added dropwise within 20 minutes at 0°C A solution of ethyl butanecarboxylate (3.80 g, 14.6 mmol) in tetrahydrofuran (22 mL). The reaction was stirred at room temperature for 2 hours. Add ethyl acetate (50mL) to dilute, and the organic phase was washed with water (30mL) and saturated brine (30mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product 3-(benzyloxy)-1- (Hydroxymethyl)cyclobutyronitrile (3.70 g, colorless oil). 1 H NMR: (400MHz, DMSO- d 6 ) δ 7.38-7.25 (m, 5H), 5.57-5.52 (m, 1H), 4.39-4.36 (m, 2H), 4.13-4.04 (m, 1H), 3.57-3.51 (m, 2H), 2.58-2.51 (m, 1H), 2.49-2.45 (m, 1H), 2.31-2.09 (m, 2H).

第四步。 the fourth step.

(3-(苄氧基)-1-氰基環丁基)甲基甲磺酸酯。 (3-(Benzyloxy)-1-cyanocyclobutyl)methanesulfonate.

將3-(苄氧基)-1-(羥甲基)環丁腈(3.70g，15.3mmol)，三乙胺(3.10g，30.6mmol)溶於二氯甲烷(35mL)中，在0℃下緩慢加入甲烷磺醯氯(3.29g，28.7mmol)。反應液室溫攪拌4小時，加入飽和氯化銨(30mL)，用乙酸乙酯(50mL x 2)萃取，有機相用無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到粗產物(3-(苄氧基)-1-氰基環丁基)甲基甲磺酸酯(4.56g，深棕色油狀)。1H NMR：(400MHz，Methonal-d 4)δ7.36-7.26(m，5H)，4.47-4.45(m，2H)，4.44-4.38(m，2H)，3.21-3.18(m，1H)，3.17-3.14(m，3H)，2.81-2.60(m，2H)，2.53-2.26(m，2H)。 Dissolve 3-(benzyloxy)-1-(hydroxymethyl)cyclobutyronitrile (3.70g, 15.3mmol), triethylamine (3.10g, 30.6mmol) in dichloromethane (35mL) at 0°C Slowly add methanesulfonyl chloride (3.29g, 28.7mmol). The reaction solution was stirred at room temperature for 4 hours, saturated ammonium chloride (30 mL) was added, and the mixture was extracted with ethyl acetate (50 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product (3-(benzyl (Oxy)-1-cyanocyclobutyl)methanesulfonate (4.56 g, dark brown oil). 1 H NMR: (400MHz, Methonal- d 4 ) δ7.36-7.26 (m, 5H), 4.47-4.45 (m, 2H), 4.44-4.38 (m, 2H), 3.21-3.18 (m, 1H), 3.17-3.14 (m, 3H), 2.81-2.60 (m, 2H), 2.53-2.26 (m, 2H).

第五步。 the fifth step.

3-(苄氧基)-1-((3，7-二甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)環丁腈。 3-(Benzyloxy)-1-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -purin-1-yl)methyl ) Cyclobutyronitrile.

將(3-(苄氧基)-1-氰基環丁基)甲基甲磺酸酯(4.50g，15.2mmol)，3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(2.75g，15.2mmol)及碘化 鉀(1.26g，7.62mmol)溶於N，N-二甲基甲醯胺(100mL)中，加入碳酸鉀(6.32g，45.7mmol)，反應120℃加熱回流4小時。反應液冷却至室溫，過濾，濾液減壓濃縮，加入水(50mL)，並用乙酸乙酯(50mL x 3)萃取。合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到粗產物3-(苄氧基)-1-((3，7-二甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)環丁腈(4.60g，黃色固體)。MS-ESI計算值[M+H]+ 380，實測值380。 Add (3-(benzyloxy)-1-cyanocyclobutyl)methanesulfonate (4.50g, 15.2mmol), 3,7-dimethyl- 1H -purine-2,6-( 3 H , 7 H )-dione (2.75g, 15.2mmol) and potassium iodide (1.26g, 7.62mmol) were dissolved in N , N -dimethylformamide (100mL), and potassium carbonate (6.32g, 45.7) mmol), the reaction was heated to reflux at 120°C for 4 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure, water (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product 3-(benzyloxy)-1-((3,7-dimethyl-2,6-dioxo-2, 3,6,7-Tetrahydro- 1H -purin-1-yl)methyl)cyclobutyronitrile (4.60 g, yellow solid). MS-ESI calculated value [M+H] + 380, measured value 380.

第六步。 The sixth step.

1-((3，7-二甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)-3-羥基環丁腈。 1-((3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -purin-1-yl)methyl)-3-hydroxycyclobutyronitrile .

將3-(苄氧基)-1-((3，7-二甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)環丁腈(100mg，0.263mmol)溶於二氯甲烷(10mL)中，並加入氯化鐵(128mg，0.790mmol)。反應室溫攪拌12小時。加入水(10mL)並用二氯甲烷(40mL x 3)萃取。合併有機相，並用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到的產品用製備高效液相色譜純化得到產物1-((3，7-二甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)-3-羥基環丁腈(12.0mg，黃色固體)，產率：16%。1H NMR：(400MHz，CDCl3)δ7.56(s，1H)，4.66-4.49(m，1H)，4.45-4.37(m，2H)，4.01(s，3H)，3.62(s，3H)，2.96-2.85(m，2H)，2.60-2.49(m，2H)。MS-ESI計算值[M+H]+ 290，實測值290。 The 3-(benzyloxy)-1-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)methan Cyclobutyronitrile (100 mg, 0.263 mmol) was dissolved in dichloromethane (10 mL), and ferric chloride (128 mg, 0.790 mmol) was added. The reaction was stirred at room temperature for 12 hours. Water (10 mL) was added and extracted with dichloromethane (40 mL x 3). The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product obtained was purified by preparative high performance liquid chromatography to obtain the product 1-((3,7-dimethyl-2,6-dioxo-2 ,3,6,7-Tetrahydro- 1H -purin-1-yl)methyl)-3-hydroxycyclobutyronitrile (12.0mg, yellow solid), yield: 16%. 1 H NMR: (400MHz, CDCl 3 ) δ7.56 (s, 1H), 4.66-4.49 (m, 1H), 4.45-4.37 (m, 2H), 4.01 (s, 3H), 3.62 (s, 3H) , 2.96-2.85 (m, 2H), 2.60-2.49 (m, 2H). MS-ESI calculated value [M+H] + 290, measured value 290.

實施例7。 Example 7.

Figure 105135042-A0305-02-0040-58
Figure 105135042-A0305-02-0040-58
Figure 105135042-A0305-02-0041-60
Figure 105135042-A0305-02-0041-60

第一步。 first step.

甲基3-(羥基甲基)雙環[1.1.1]戊烷-1-羧酸甲酯。 Methyl 3-(hydroxymethyl)bicyclo[1.1.1]pentane-1-carboxylic acid methyl ester.

將3-(甲氧羰基)雙環[1.1.1]戊烷-1-羧酸(100mg，0.587mmol)和三乙胺(71.0mg，0.705mmol)溶於四氫呋喃(20mL)中，-10℃條件下緩慢滴加氯甲酸甲酯(56.0mg，0.587mmol)。反應液於0℃條件下攪拌半小時，然後加入硼氫化鈉(33.0mg，0.881mmol)，繼續反應2小時。反應液中加入水(10mL)，乙酸乙酯(10mL x 3)萃取，合併有機相並用飽和氯化鈉(10mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到甲基3-(羥基甲基)雙環[1.1.1]戊烷-1-羧酸甲酯(80.0mg，無色油狀物)，產率：87%。1H NMR：(400MHz，CDCl3)δ3.65(s，3H)，3.60(s，2H)，2.00(s，6H)。 Dissolve 3-(methoxycarbonyl)bicyclo[1.1.1]pentane-1-carboxylic acid (100mg, 0.587mmol) and triethylamine (71.0mg, 0.705mmol) in tetrahydrofuran (20mL) at -10℃ Methyl chloroformate (56.0 mg, 0.587 mmol) was slowly added dropwise. The reaction solution was stirred at 0°C for half an hour, then sodium borohydride (33.0 mg, 0.881 mmol) was added, and the reaction was continued for 2 hours. Water (10mL) was added to the reaction solution, extracted with ethyl acetate (10mL x 3), the organic phases were combined and washed with saturated sodium chloride (10mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain methyl 3- (Hydroxymethyl)bicyclo[1.1.1]pentane-1-carboxylic acid methyl ester (80.0 mg, colorless oil), yield: 87%. 1 H NMR: (400 MHz, CDCl 3 ) δ 3.65 (s, 3H), 3.60 (s, 2H), 2.00 (s, 6H).

第二步。 The second step.

甲基3-(((甲基磺醯基)氧基)甲基)雙環[1.1.1]戊烷-1-羧酸甲酯。 Methyl 3-(((methylsulfonyl)oxy)methyl)bicyclo[1.1.1]pentane-1-carboxylic acid methyl ester.

將甲基3-(羥基甲基)雙環[1.1.1]戊烷-1-羧酸甲酯(40.0mg，0.256mmol)和三乙胺(39.0mg，0.384mmol)溶於二氯甲烷(15mL)中，0℃條件下緩慢滴加甲烷磺醯氯(35.0mg，0.307mmol)。反應液於0℃條件下攪拌2小時，反應液中加入二氯甲烷(10mL)稀釋，有機相用水(10mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到甲基3-(((甲基磺醯基)氧基)甲基)雙環[1.1.1]戊烷-1-羧酸甲酯(50.0mg，黃色油狀物)，產率：83%。 Methyl 3-(hydroxymethyl)bicyclo[1.1.1]pentane-1-carboxylic acid methyl ester (40.0mg, 0.256mmol) and triethylamine (39.0mg, 0.384mmol) were dissolved in dichloromethane (15mL ), slowly dropwise add methanesulfonyl chloride (35.0mg, 0.307mmol) at 0°C. The reaction solution was stirred at 0°C for 2 hours. Dichloromethane (10mL) was added to the reaction solution for dilution. The organic phase was washed with water (10mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain methyl 3-( ((Methylsulfonyl)oxy)methyl)bicyclo[1.1.1]pentane-1-carboxylic acid methyl ester (50.0 mg, yellow oil), yield: 83%.

第三步。 third step.

甲基3-((3，7-二甲基-2，6-二氧-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)雙環[1.1.1]戊烷-1-羧酸甲酯。 Methyl 3-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)methyl)bicyclo[1.1.1] Methyl pentane-1-carboxylate.

將甲基3-(((甲基磺醯基)氧基)甲基)雙環[1.1.1]戊烷-1-羧酸甲酯(100mg，0.426mmol)和3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(77.0mg，0.427mmol)溶於N，N-二甲基甲醯胺(20mL)中，室溫條件下加入碳酸鉀(88.0mg，0.640mmol)和碘化鉀(8.00mg，0.0430mmol)。反應液於100℃條件下攪拌2小時，反應液冷却至室溫濃縮，加入乙酸乙酯(20mL)稀釋，有機相用水(20mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱層析法分離純化(1：1石油醚/乙酸乙酯，Rf=0.2)得到甲基3-((3，7-二甲基-2，6-二氧-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)雙環[1.1.1]戊烷-1-羧酸甲酯(100mg，黃色固體)，產率：73%。1H NMR：(400MHz，CDCl3)δ7.50(s，1H)，4.12(s，2H)，3.95(s，3H)，3.60(s，3H)，3.53(s，3H)，1.95(s，6H)。MS-ESI計算值[M+H]+ 319，實測值319。 Methyl 3-(((methylsulfonyl)oxy)methyl)bicyclo[1.1.1]pentane-1-carboxylic acid methyl ester (100mg, 0.426mmol) and 3,7-dimethyl- 1 H -purine-2,6-(3 H , 7 H )-dione (77.0mg, 0.427mmol) was dissolved in N,N -dimethylformamide (20mL), and potassium carbonate was added at room temperature (88.0 mg, 0.640 mmol) and potassium iodide (8.00 mg, 0.0430 mmol). The reaction solution was stirred at 100°C for 2 hours. The reaction solution was cooled to room temperature and concentrated, diluted with ethyl acetate (20mL), the organic phase was washed with water (20mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Separated and purified by silica gel column chromatography (1:1 petroleum ether/ethyl acetate, Rf=0.2) to obtain methyl 3-((3,7-dimethyl-2,6-dioxo-2,3,6 , 7-Tetrahydro- 1H -purin-1-yl)methyl)bicyclo[1.1.1]pentane-1-carboxylic acid methyl ester (100mg, yellow solid), yield: 73%. 1 H NMR: (400MHz, CDCl 3 ) δ 7.50 (s, 1H), 4.12 (s, 2H), 3.95 (s, 3H), 3.60 (s, 3H), 3.53 (s, 3H), 1.95 (s , 6H). MS-ESI calculated value [M+H] + 319, measured value 319.

第四步。 the fourth step.

3-((3，7-二甲基-2，6-二氧-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)雙環[1.1.1]戊烷-1-羧酸。 3-((3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)methyl)bicyclo[1.1.1]pentane -1-carboxylic acid.

將甲基3-((3，7-二甲基-2，6-二氧-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)雙環[1.1.1]戊烷-1-羧酸甲酯(100mg，0.314mmol)溶於四氫呋喃(15mL)和水(5mL)中，室溫條件下加入氫氧化鋰(26.0mg，0.628mmol)。室溫攪拌2小時後，反應液加入2N稀鹽酸(10mL)調PH值至4，乙酸乙酯(15mL x 3)萃取，合併有機相用飽和氯化鈉溶液(20mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮3-((3，7-二甲基-2，6-二氧-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)雙環[1.1.1]戊烷-1-羧酸(90.0mg，白色固體)，產率：94%。 The methyl 3-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)methyl)bicyclo[1.1.1 ] Methyl pentane-1-carboxylate (100 mg, 0.314 mmol) was dissolved in tetrahydrofuran (15 mL) and water (5 mL), and lithium hydroxide (26.0 mg, 0.628 mmol) was added at room temperature. After stirring for 2 hours at room temperature, the reaction solution was added with 2N dilute hydrochloric acid (10mL) to adjust the pH to 4, extracted with ethyl acetate (15mL x 3), and the combined organic phases were washed with saturated sodium chloride solution (20mL x 2), anhydrous sulfuric acid The sodium was dried, filtered, and the filtrate was concentrated under reduced pressure. 3-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)methyl ) Bicyclo[1.1.1]pentane-1-carboxylic acid (90.0 mg, white solid), yield: 94%.

MS-ESI計算值[M+H]+ 305，實測值305。 MS-ESI calculated value [M+H] + 305, measured value 305.

第五步。 the fifth step.

3-((3，7-二甲基-2，6-二氧-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)-N-甲氧基-N-甲基雙環[1.1.1]戊烷-1-醯胺。 3-((3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -purin-1-yl)methyl) -N -methoxy- N -Methylbicyclo[1.1.1]pentane-1-amide.

將3-((3，7-二甲基-2，6-二氧-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)雙環[1.1.1]戊烷-1-甲酸(30.0mg，0.0986mmol)和N，O-二甲基羥胺(10.0mg，0.0986mmol)溶於二氯甲烷(20mL)中，室溫條件下加入2-(7-偶氮苯並三氮唑)-N，N，N’，N’-四甲基脲六氟磷酸酯(75.0mg，0.197mmol)和二異丙基乙胺(19.0mg，0.148mmol)。室溫攪拌12小時後，反應液加入水(20mL)，二氯甲烷(20mL x 2)萃取，合併有機相用飽和氯化銨溶液(20mL x 2)洗滌。無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱層析法分離純化(1：2石油醚/乙酸乙酯，Rf=0.2)得到3-((3，7-二甲基-2，6-二氧-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)-N-甲氧基-N-甲基雙環[1.1.1]戊烷-1-醯胺(30.0mg，白色固體)，產率：88%。 The 3-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)methyl)bicyclo[1.1.1]pentan Alkane-1-carboxylic acid (30.0mg, 0.0986mmol) and N , O -dimethylhydroxylamine (10.0mg, 0.0986mmol) were dissolved in dichloromethane (20mL), and 2-(7-azo Benzotriazole) -N , N , N ', N' -tetramethylurea hexafluorophosphate (75.0 mg, 0.197 mmol) and diisopropylethylamine (19.0 mg, 0.148 mmol). After stirring for 12 hours at room temperature, the reaction solution was added with water (20 mL), extracted with dichloromethane (20 mL x 2), and the combined organic phases were washed with saturated ammonium chloride solution (20 mL x 2). Dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and separate and purify by silica gel column chromatography (1:2 petroleum ether/ethyl acetate, Rf=0.2) to obtain 3-((3,7-dimethyl-2, 6-Dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)methyl) -N -methoxy- N -methylbicyclo[1.1.1]pentane-1- Amide (30.0 mg, white solid), yield: 88%.

MS-ESI計算值[M+H]+ 348，實測值348。 MS-ESI calculated value [M+H] + 348, measured value 348.

第六步。 The sixth step.

1-((3-乙醯基雙環[1.1.1]戊烷-1-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((3-Acetylbicyclo[1.1.1]pentan-1-yl)methyl)-3,7-dimethyl-1 H -purine-2,6-(3 H ,7 H ) -Diketone.

將3-((3，7-二甲基-2，6-二氧-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)-N-甲氧基-N-甲基雙環[1.1.1]戊烷-1-醯胺(20.0mg，0.0575mmol)溶於四氫呋喃(20mL)中，反應液於-78℃條件下加入甲基溴化鎂(3M乙醚溶液，0.040mL，0.120mmol)，繼續攪拌30分鐘後升至室溫反應4小時。反應液於0℃條件下加入飽和氯化銨溶液(10mL)，乙酸乙酯(15mL x 3)萃取，合併有機相用飽和氯化鈉溶液(20mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用矽膠柱層析法分離純化(1：1石油醚/乙酸乙酯，Rf=0.5)得到1-((3-乙醯基雙環[1.1.1]戊烷-1-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(15.0mg，無色油狀物)，產率：86%。1H NMR：(400MHz，CDCl3)δ7.55(s，1H)，4.17(s，2H)，3.99(s，3H)，3.59(s，3H)，2.07(s，3H)，1.97(s，6H)。MS-ESI計算值[M+H]+ 303，實測值303。 The 3-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)methyl) -N -methoxy- N -methylbicyclo[1.1.1]pentane-1-amide (20.0mg, 0.0575mmol) was dissolved in tetrahydrofuran (20mL), and the reaction solution was added with methylmagnesium bromide (3M ether solution at -78℃) , 0.040mL, 0.120mmol), continue to stir for 30 minutes and then rise to room temperature to react for 4 hours. The reaction solution was added with saturated ammonium chloride solution (10mL) at 0℃, extracted with ethyl acetate (15mL x 3), the combined organic phases were washed with saturated sodium chloride solution (20mL x 2), dried with anhydrous sodium sulfate, filtered, The filtrate was concentrated under reduced pressure. Separated and purified by silica gel column chromatography (1:1 petroleum ether/ethyl acetate, Rf=0.5) to obtain 1-((3-acetylbicyclo[1.1.1]pentane-1-yl)methyl)- 3,7-dimethyl -1 H - purine -2,6- (3 H, 7 H) - dione (15.0 mg, colorless oil). yield: 86%. 1 H NMR: (400MHz, CDCl 3 ) δ 7.55 (s, 1H), 4.17 (s, 2H), 3.99 (s, 3H), 3.59 (s, 3H), 2.07 (s, 3H), 1.97 (s , 6H). MS-ESI calculated value [M+H] + 303, measured value 303.

第七步。 The seventh step.

3，7-二甲基-1-((3-(1，1，1-三氟-2-羥基丙烷-2-基)雙環[1.1.1]戊烷-1-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3,7-Dimethyl-1-((3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)bicyclo[1.1.1]pentan-1-yl)methyl)- 1 H -purine-2,6-(3 H ,7 H )-dione.

將1-((3-乙醯基雙環[1.1.1]戊烷-1-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(20.0mg，0.0660mmol)和三氟甲基三甲基氯矽烷(19.0mg，0.132mmol)溶於四氫呋喃(15mL)中，反應液於室溫條件下加入氟化銫(10.0mg，00660mmol)，室溫繼續反應12小時。反應液中加入2N稀鹽酸(10mL)，攪拌30分鐘，乙酸乙酯(20mL x 2)萃 取，合併有機相用飽和碳酸氫鈉溶液(20mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用高效液相色譜純化得3，7-二甲基-1-((3-(1，1，1-三氟-2-羥基丙烷-2-基)雙環[1.1.1]戊烷-1-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮(5.00mg，無色油狀物)，產率：20%。1H NMR：(400MHz，CDCl3)δ7.59(s，1H)，4.20(s，2H)，4.02(s，3H)，3.59(s，3H)，1.97(s，6H)，1.79(s，3H)。MS-ESI計算值[M+H]+ 373，實測值373。 1-((3-Acetylbicyclo[1.1.1]pentan-1-yl)methyl)-3,7-dimethyl- 1H -purine-2,6-( 3H , 7H )-Diketone (20.0mg, 0.0660mmol) and trifluoromethyltrimethylchlorosilane (19.0mg, 0.132mmol) were dissolved in tetrahydrofuran (15mL), the reaction solution was added with cesium fluoride (10.0mg , 00660mmol), and continue the reaction at room temperature for 12 hours. Add 2N dilute hydrochloric acid (10mL) to the reaction solution, stir for 30 minutes, and extract with ethyl acetate (20mL x 2). The combined organic phases are washed with saturated sodium bicarbonate solution (20mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate is reduced. Concentrated by pressure and purified by high performance liquid chromatography to obtain 3,7-dimethyl-1-((3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)bicyclo[1.1.1]pentan 1-yl) methyl) -1 H - purine -2,6- (3 H, 7 H) - dione (5.00mg, colorless oil). yield: 20%. 1 H NMR: (400MHz, CDCl 3 ) δ 7.59 (s, 1H), 4.20 (s, 2H), 4.02 (s, 3H), 3.59 (s, 3H), 1.97 (s, 6H), 1.79 (s) , 3H). MS-ESI calculated value [M+H] + 373, measured value 373.

實施例8。 Example 8.

Figure 105135042-A0305-02-0045-61
Figure 105135042-A0305-02-0045-61

第一步 first step

3，7-二甲基-1-[[3-[2，2，2-三氟-1-羥基-1-(三氟甲基)乙基]環丁基]甲基]嘌呤-2，6-二酮。 3,7-Dimethyl-1-[[3-[2,2,2-Trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]cyclobutyl]methyl]purine-2, 6-Diketone.

將3，7-二甲基-1-[[3-(2，2，2-三氟-1，1-二羥基-乙基)環丁基]甲基]嘌呤-2，6-二酮(60.0mg，0.165mmol)，氟化銫(25.2mg，0.165mmol)溶於四氫呋喃(10mL)中，室溫下加入三氟甲基三甲基氯矽烷(70.6mg，0.496mmol)，攪拌12小時。加入水(20mL)淬滅反應。用乙酸乙酯萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用製備型高效液相色譜純化，得到產物1(8.00mg，黃色固體)(異構體1，第一個峰)，產率：12%。1H NMR：(400MHz，Methonal-d 4 )δ8.01(s，1H)，4.22-4.17(m，2H)，4.01(s，3H)，3.54(s，3H)， 3.55-3.19(m，1H)，2.63-2.56(m，1H)，2.50-2.42(m，2H)，1.82-1.78(m，2H)。MS-ESI計算值[M+H]+ 415，實測值415。 3,7-Dimethyl-1-[[3-(2,2,2-trifluoro-1,1-dihydroxy-ethyl)cyclobutyl]methyl]purine-2,6-dione (60.0mg, 0.165mmol), cesium fluoride (25.2mg, 0.165mmol) was dissolved in tetrahydrofuran (10mL), added trifluoromethyltrimethylchlorosilane (70.6mg, 0.496mmol) at room temperature, and stirred for 12 hours . The reaction was quenched by adding water (20 mL). Extract with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purified by preparative high performance liquid chromatography to obtain product 1 (8.00 mg, yellow solid) (isomer 1, first peak), yield: 12%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 8.01 (s, 1H), 4.22-4.17 (m, 2H), 4.01 (s, 3H), 3.54 (s, 3H), 3.55-3.19 (m, 1H), 2.63-2.56 (m, 1H), 2.50-2.42 (m, 2H), 1.82-1.78 (m, 2H). MS-ESI calculated value [M+H] + 415, measured value 415.

以及產物2(異構體2，第二個峰)，產率：6%。1H NMR：(400MHz，Methonal-d 4 )δ8.20(s，1H)，4.04-4.00(m，5H)，3.55(s，3H)2.70-2.65(m，1H)，2.55-2.53(m，1H)，2.17-2.12(m，2H)，2.02-1.98(m，2H)。MS-ESI計算值[M+H]+ 415，實測值415。 And product 2 (isomer 2, second peak), yield: 6%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 8.20 (s, 1H), 4.04-4.00 (m, 5H), 3.55 (s, 3H) 2.70-2.65 (m, 1H), 2.55-2.53 (m , 1H), 2.17-2.12 (m, 2H), 2.02-1.98 (m, 2H). MS-ESI calculated value [M+H] + 415, measured value 415.

實施例9。 Example 9.

Figure 105135042-A0305-02-0046-62
Figure 105135042-A0305-02-0046-62

第一步。 first step.

3-亞甲基環丁烷羧酸。 3-methylenecyclobutane carboxylic acid.

將3-亞甲基環丁腈(10.0g，107mmol)和氫氧化鉀(18.1g，322mmol)溶於乙醇(100mL)和水(50mL)中，100℃下反應2小時後。加入1N鹽酸(120mL)。二氯甲烷萃取(30mL x 3)，無水硫酸鈉乾燥， 過濾，濾液減壓濃縮得到3-亞甲基環丁烷羧酸(11.0g，黃色油狀)，產率：91%。1H NMR：(400MHz，Methonal-d 4 )δ4.83-4.76(m，2H)，3.15-2.96(m，1H)，2.95-2.92(m，4H)。 3-Methylene cyclobutyronitrile (10.0 g, 107 mmol) and potassium hydroxide (18.1 g, 322 mmol) were dissolved in ethanol (100 mL) and water (50 mL) and reacted at 100°C for 2 hours. 1N hydrochloric acid (120 mL) was added. Extraction with dichloromethane (30 mL x 3), drying with anhydrous sodium sulfate, filtration, and concentration of the filtrate under reduced pressure to obtain 3-methylenecyclobutane carboxylic acid (11.0 g, yellow oil), yield: 91%. 1 H NMR: (400 MHz, Methonal- d 4 ) δ 4.83-4.76 (m, 2H), 3.15 to 2.96 (m, 1H), 2.95 to 2.92 (m, 4H).

第二步。 The second step.

甲基-3-亞甲基環丁烷羧酸。 Methyl-3-methylenecyclobutane carboxylic acid.

將3-亞甲基環丁烷羧酸(11.0g，98.1mmol)和碳酸鉀(27.1g，196mmol)溶於丙酮(100mL)中，25℃下加入硫酸二甲酯(14.8g，117mmol)，70℃反應12小時後。加入水(20mL)淬滅反應，二氯甲烷萃取(30mL x 3)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到甲基-3-亞甲基環丁烷羧酸(12.0g，黃色油狀)，產率：97%。1H NMR：(400MHz，Methonal-d 4 )δ4.83-4.79(m，2H)，3.96(s，2H)，3.68(s，3H)，3.17-3.15(m，1H)，2.95-2.92(m，2H)。 Dissolve 3-methylenecyclobutanecarboxylic acid (11.0g, 98.1mmol) and potassium carbonate (27.1g, 196mmol) in acetone (100mL), add dimethyl sulfate (14.8g, 117mmol) at 25°C, After reacting at 70°C for 12 hours. The reaction was quenched by adding water (20mL), extracted with dichloromethane (30mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain methyl-3-methylenecyclobutanecarboxylic acid (12.0g, yellow oil) State), yield: 97%. 1 H NMR: (400MHz, Methonal- d 4 )δ4.83-4.79(m, 2H), 3.96(s, 2H), 3.68(s, 3H), 3.17-3.15(m, 1H), 2.95-2.92( m, 2H).

第三步。 third step.

甲基3-(羥甲基)環丁烷羧酸乙酯。 Ethyl methyl 3-(hydroxymethyl)cyclobutanecarboxylate.

將甲基-3-亞甲基環丁烷羧酸(2.00g，15.8mmol)溶於四氫呋喃(30mL)中，-10℃下滴加硼烷二甲硫醚(3.61g，47.5mmol)，然後-10℃反應3小時，加入3N氫氧化鈉水溶液(10mL)和雙氧水(5mL)，繼續反應1小時，反應液加入飽和硫代硫酸鈉水溶液(30mL)淬滅反應，二氯甲烷萃取(10mL x 3)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到甲基3-(羥甲基)環丁烷羧酸乙酯(2.00g，黃色油狀)，產率：87%。1H NMR：(400MHz，Methonal-d 4 )δ3.70(s，3H)，3.58(d，J=6.8Hz，1H)，3.49(d，J=6.8Hz，1H)，3.10-3.05(m，1H)，2.32-2.26(m，3H)，2.03-1.98(m，2H)。 Dissolve methyl-3-methylenecyclobutanecarboxylic acid (2.00g, 15.8mmol) in tetrahydrofuran (30mL), add borane dimethyl sulfide (3.61g, 47.5mmol) dropwise at -10°C, then React at -10°C for 3 hours, add 3N sodium hydroxide aqueous solution (10mL) and hydrogen peroxide (5mL), continue the reaction for 1 hour, add saturated sodium thiosulfate aqueous solution (30mL) to quench the reaction, and extract with dichloromethane (10mL x 3) Drying with anhydrous sodium sulfate, filtering, and concentrating the filtrate under reduced pressure to obtain ethyl methyl 3-(hydroxymethyl)cyclobutanecarboxylate (2.00 g, yellow oil), yield: 87%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 3.70 (s, 3H), 3.58 (d, J = 6.8 Hz, 1H), 3.49 (d, J = 6.8 Hz, 1H), 3.10-3.05 (m , 1H), 2.32-2.26 (m, 3H), 2.03-1.98 (m, 2H).

第四步。 the fourth step.

3-(甲基磺醯氧基甲基)環丁烷羧酸乙酯。 Ethyl 3-(methylsulfonyloxymethyl)cyclobutanecarboxylate.

將3-(羥甲基)環丁烷羧酸乙酯(1.00g，6.94mmol)和三乙胺(2.11g，20.8mmol)溶於二氯甲烷(20mL)中，0℃下加入甲烷磺醯氯(1.59g，13.9mmol)。反應液緩慢升至室溫，攪拌2小時。加入碳酸氫鈉水溶液(50mL)淬滅反應。用二氯甲烷萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到3-(甲基磺醯氧基甲基)環丁烷羧酸乙酯(1.40g，黃色油狀)，產率：91%。1H NMR：(400MHz，Methonal-d 4 )δ4.28(d，J=6.8Hz，1H)，4.19(d，J=6.8Hz，1H)，3.70(s，3H)，3.20-3.08(m，4H)，2.40-2.34(m，3H)，2.13-2.09(m，2H)。MS-ESI計算值[M+H]+ 223，實測值223。 Dissolve ethyl 3-(hydroxymethyl)cyclobutanecarboxylate (1.00g, 6.94mmol) and triethylamine (2.11g, 20.8mmol) in dichloromethane (20mL), add methanesulfonate at 0°C Chlorine (1.59 g, 13.9 mmol). The reaction solution was slowly raised to room temperature and stirred for 2 hours. The reaction was quenched by the addition of aqueous sodium bicarbonate (50 mL). Extract with dichloromethane (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain ethyl 3-(methylsulfonyloxymethyl)cyclobutanecarboxylate (1.40g, yellow oily) , Yield: 91%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 4.28 (d, J = 6.8 Hz, 1H), 4.19 (d, J = 6.8 Hz, 1H), 3.70 (s, 3H), 3.20-3.08 (m , 4H), 2.40-2.34 (m, 3H), 2.13-2.09 (m, 2H). MS-ESI calculated value [M+H] + 223, measured value 223.

第五步。 the fifth step.

3-[(3，7-二甲基-2，6-二氧代-嘌呤-1-基)甲基]環丁烷羧酸乙酯 3-[(3,7-Dimethyl-2,6-dioxo-purin-1-yl)methyl]cyclobutanecarboxylic acid ethyl ester

將3-(甲基磺醯氧基甲基)環丁烷羧酸乙酯(1.40g，6.30mmol)，3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(1.13g，6.30mmol)，碘化鉀(209mg，1.26mmol)和碳酸鉀(2.61g，18.90mmol)溶於N，N-二甲基甲醯胺(100mL)中。反應液升溫至120℃，攪拌3小時。冷却至室溫，過濾，加入水(100mL)，用二氯甲烷萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到3-[(3，7-二甲基-2，6-二氧代-嘌呤-1-基)甲基]環丁烷羧酸乙酯(1.50g，黃色固體)，產率：78%。 The ethyl 3-(methylsulfonyloxymethyl)cyclobutanecarboxylate (1.40g, 6.30mmol), 3,7-dimethyl- 1H -purine-2,6-( 3H ,7 H )-diketone (1.13 g, 6.30 mmol), potassium iodide (209 mg, 1.26 mmol) and potassium carbonate (2.61 g, 18.90 mmol) were dissolved in N , N -dimethylformamide (100 mL). The reaction solution was heated to 120°C and stirred for 3 hours. Cool to room temperature, filter, add water (100 mL), and extract with dichloromethane (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 3-[(3,7-dimethyl-2,6-dioxo-purin-1-yl)methan Cyclobutane carboxylate (1.50 g, yellow solid), yield: 78%.

1H NMR：(400MHz，Methonal-d 4 )δ7.51(s，1H)，4.18-4.10(m，2H)，3.99(s，3H)，3.67(s，3H)，3.55(s，3H)，3.26-2.65(m，2H)，2.29-2.13(m，4H)。MS-ESI計算值[M+H]+ 307，實測值307。 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.51 (s, 1H), 4.18-4.10 (m, 2H), 3.99 (s, 3H), 3.67 (s, 3H), 3.55 (s, 3H) , 3.26-2.65 (m, 2H), 2.29-2.13 (m, 4H). MS-ESI calculated value [M+H] + 307, measured value 307.

第六步。 The sixth step.

3-[(3，7-二甲基-2，6-二氧代-嘌呤-1-基)甲基]環丁烷羧酸 3-[(3,7-Dimethyl-2,6-dioxo-purin-1-yl)methyl]cyclobutane carboxylic acid

將3-[(3，7-二甲基-2，6-二氧代-嘌呤-1-基)甲基]環丁烷羧酸乙酯(1.00g，3.26mmol)，氫氧化鉀(548mg，9.78mmol)溶於甲醇(10mL)和水(5mL)中。反應液升溫至90℃，攪拌3小時。冷却至室溫，加入1N鹽酸(20mL)中和過濾，用二氯甲烷萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到3-[(3，7-二甲基-2，6-二氧代-嘌呤-1-基)甲基]環丁烷羧酸(800.00mg，黃色固體)，產率：84%。MS-ESI計算值[M+H]+ 293，實測值293。 Ethyl 3-[(3,7-dimethyl-2,6-dioxo-purin-1-yl)methyl]cyclobutanecarboxylate (1.00g, 3.26mmol), potassium hydroxide (548mg , 9.78mmol) was dissolved in methanol (10mL) and water (5mL). The reaction solution was heated to 90°C and stirred for 3 hours. Cool to room temperature, add 1N hydrochloric acid (20 mL) to neutralize and filter, and extract with dichloromethane (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 3-[(3,7-dimethyl-2,6-dioxo-purin-1-yl)methan Cyclobutane carboxylic acid (800.00 mg, yellow solid), yield: 84%. MS-ESI calculated value [M+H] + 293, measured value 293.

第七步。 The seventh step.

3-[(3，7-二甲基-2，6-二氧代-嘌呤-1-基)甲基]-N-甲氧基-N-甲基環丁烷甲醯胺。 3-[(3,7-Dimethyl-2,6-dioxo-purin-1-yl)methyl] -N -methoxy- N -methylcyclobutanecarboxamide.

將3-[(3，7-二甲基-2，6-二氧代-嘌呤-1-基)甲基]環丁烷羧酸(300mg，1.03mmol)，N，O-二甲基羥胺鹽酸鹽(200mg，2.05mmol)，1-乙基-(3-二甲基氨基丙基)碳二亞胺鹽酸鹽(394mg，2.06mmol)，1-羥基苯並三唑(27.8mg，0.206mmol)和三乙胺(312mg，3.09mmol)溶於二氯甲烷(10mL)中。25℃攪拌12小時。反應液減壓濃縮，用製備TLC板分離純化(乙酸乙酯，Rf值=0.3)得到3-[(3，7-二甲基-2，6-二氧代-嘌呤-1-基)甲基]-N-甲氧基-N-甲基環丁烷甲醯胺(200mg，黃色固體)，產率：58%。MS-ESI計算值[M+H]+ 336，實測值336。 Add 3-[(3,7-dimethyl-2,6-dioxo-purin-1-yl)methyl]cyclobutanecarboxylic acid (300mg, 1.03mmol), N , O -dimethylhydroxylamine Hydrochloride (200mg, 2.05mmol), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (394mg, 2.06mmol), 1-hydroxybenzotriazole (27.8mg, 0.206 mmol) and triethylamine (312 mg, 3.09 mmol) were dissolved in dichloromethane (10 mL). Stir at 25°C for 12 hours. The reaction solution was concentrated under reduced pressure, and separated and purified with a preparative TLC plate (ethyl acetate, Rf value=0.3) to obtain 3-[(3,7-dimethyl-2,6-dioxo-purin-1-yl)methan Yl] -N -methoxy- N -methylcyclobutanecarboxamide (200 mg, yellow solid), yield: 58%. MS-ESI calculated value [M+H] + 336, measured value 336.

第八步。 The eighth step.

1-[(3-乙醯基環丁基)甲基]-3，7-二甲基嘌呤-2，6-二酮 1-[(3-Acetylcyclobutyl)methyl]-3,7-dimethylpurine-2,6-dione

將3-[(3，7-二甲基-2，6-二氧代-嘌呤-1-基)甲基]-N-甲氧基-N-甲基環丁烷甲醯胺(300mg，0.894mmol)溶於四氫呋喃(10mL)中。0℃下滴加甲基溴化鎂(3M乙醚溶液，1.49mL，4.47mmol)攪拌3小時。反 應液加入飽和氯化銨溶液(20mL)淬滅反應，用二氯甲烷萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用製備TLC板分離純化(乙酸乙酯，Rf值=0.5)得到1-[(3-乙醯基環丁基)甲基]-3，7-二甲基嘌呤-2，6-二酮(200mg，黃色固體)，產率：77%。 Add 3-[(3,7-dimethyl-2,6-dioxo-purin-1-yl)methyl] -N -methoxy- N -methylcyclobutanecarboxamide (300mg, 0.894 mmol) was dissolved in tetrahydrofuran (10 mL). Methylmagnesium bromide (3M ether solution, 1.49mL, 4.47mmol) was added dropwise at 0°C and stirred for 3 hours. The reaction solution was quenched by adding saturated ammonium chloride solution (20 mL), and extracted with dichloromethane (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and separated and purified with a preparative TLC plate (ethyl acetate, Rf value = 0.5) to obtain 1-[(3-acetylcyclobutane) (Yl)methyl]-3,7-dimethylpurine-2,6-dione (200 mg, yellow solid), yield: 77%.

MS-ESI計算值[M+H]+ 291，實測值291。 MS-ESI calculated value [M+H] + 291, measured value 291.

第九步。 The ninth step.

將1-[(3-乙醯基環丁基)甲基]-3，7-二甲基嘌呤-2，6-二酮(250mg，0.861mmol)，氟化銫(130mg，0.861mmol)溶於四氫呋喃(10mL)中，室溫下加入三甲基-三氟甲基-矽烷(244mg，1.72mmol)，攪拌12小時。加入水(20mL)淬滅反應。用乙酸乙酯萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用製備型高效液相色譜純化，得到產物1(65.0mg，黃色固體)(異構體1，第一個峰)，產率：20%。1H NMR：(400MHz，Methonal-d 4 )δ8.21(s，1H)，4.23(d，J=7.6Hz，2H)，4.03(s，3H)，3.55(s，3H)，3.26-3.19(m，2H)，2.63-2.56(m，2H)，2.55-2.42(m，2H)，1.82-1.78(m，3H)。MS-ESI計算值[M+H]+ 361，實測值361。 Dissolve 1-[(3-acetylcyclobutyl)methyl]-3,7-dimethylpurine-2,6-dione (250mg, 0.861mmol), cesium fluoride (130mg, 0.861mmol) In tetrahydrofuran (10 mL), trimethyl-trifluoromethyl-silane (244 mg, 1.72 mmol) was added at room temperature, and the mixture was stirred for 12 hours. The reaction was quenched by adding water (20 mL). Extract with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purified by preparative high performance liquid chromatography to obtain product 1 (65.0 mg, yellow solid) (isomer 1, first peak), yield: 20%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 8.21 (s, 1H), 4.23 (d, J = 7.6 Hz, 2H), 4.03 (s, 3H), 3.55 (s, 3H), 3.26 to 3.19 (m, 2H), 2.63-2.56 (m, 2H), 2.55-2.42 (m, 2H), 1.82-1.78 (m, 3H). MS-ESI calculated value [M+H] + 361, measured value 361.

產物2(異構體2，第二個峰)，產率：22%。1H NMR：(400MHz，Methonal-d4)δ8.05(s，1H)，4.01-3.99(m，5H)，4.03(s，3H)，3.54(s，3H)，2.71-2.66(m，1H)，2.55-2.54(m，1H)，2.17-2.12(m，2H)，2.02-1.98(m，2H)。MS-ESI計算值[M+H]+ 361，實測值361。 Product 2 (isomer 2, second peak), yield: 22%. 1H NMR: (400MHz, Methonal-d4) δ 8.05 (s, 1H), 4.01-3.99 (m, 5H), 4.03 (s, 3H), 3.54 (s, 3H), 2.71-2.66 (m, 1H) , 2.55-2.54 (m, 1H), 2.17-2.12 (m, 2H), 2.02-1.98 (m, 2H). MS-ESI calculated value [M+H] + 361, measured value 361.

實施例10。 Example 10.

Figure 105135042-A0305-02-0050-63
Figure 105135042-A0305-02-0050-63
Figure 105135042-A0305-02-0051-64
Figure 105135042-A0305-02-0051-64

第一步。 first step.

1，4-二氧雜螺[4.4]壬烷-7-羧酸甲酯。 1,4-Dioxaspiro[4.4]nonane-7-carboxylic acid methyl ester.

將3-氧代-環戊羧酸甲酯(16.0g，110mmol)，對甲苯磺酸(14.0g，220mmol)和乙二醇(969mg，5.60mmol)溶於無水甲苯(160mL)中，加分水器後加熱回流4小時。加入水(200mL)淬滅反應，用乙酸乙酯萃取，合併有機相。合併有機相，依次用水(200mL x 2)，飽和氯化鈉溶液(200mL x 2)洗滌，用無水硫酸鎂乾燥，過濾。濾液減壓濃縮，用矽膠柱色譜法純化(5：1石油醚/乙酸乙酯，Rf=0.3)所得物1，4-二氧雜螺[4.4]壬烷-7-羧酸甲酯(6.20g，黃色油狀)，產率：29%。1H NMR：(400MHz，CDCl3)δ3.93-3.89(m，4H)，3.69(s，3H)，2.91-2.89(m，1H)，2.11-1.82(m，6H)。MS-ESI計算值[M+H]+ 187，實測值187。 Dissolve methyl 3-oxo-cyclopentacarboxylate (16.0g, 110mmol), p-toluenesulfonic acid (14.0g, 220mmol) and ethylene glycol (969mg, 5.60mmol) in dry toluene (160mL), add points After the water tank, heat to reflux for 4 hours. The reaction was quenched by adding water (200 mL), extracted with ethyl acetate, and the organic phases were combined. The organic phases were combined, washed sequentially with water (200 mL x 2), saturated sodium chloride solution (200 mL x 2), dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (5:1 petroleum ether/ethyl acetate, Rf=0.3). The resulting product 1,4-dioxaspiro[4.4]nonane-7-carboxylic acid methyl ester (6.20) g, yellow oil), yield: 29%. 1 H NMR: (400MHz, CDCl 3 ) δ 3.93-3.89 (m, 4H), 3.69 (s, 3H), 2.91-2.89 (m, 1H), 2.11-1.82 (m, 6H). MS-ESI calculated value [M+H] + 187, measured value 187.

第二步。 The second step.

(1，4-二氧雜螺[4.4]壬烷-7-基)-甲醇。 (1,4-Dioxaspiro[4.4]nonane-7-yl)-methanol.

將1，4-二氧雜螺[4.4]壬烷-7-羧酸甲酯(1.00g，10.7mmol)溶於無水四氫呋喃(30mL)中，氮氣保護，-10℃緩慢加入四氫鋁鋰(531mg， 13.9mmol)。反應液緩慢升至25℃，攪拌3小時。向反應液中依次加入水(0.5mL)，15%氫氧化鈉溶液(0.5mL)，水(1.5mL)。過濾除去不溶物，濾液減壓濃縮得到(1，4-二氧雜螺[4.4]壬烷-7-基)-甲醇(1.5mg，黃色油狀)，產率：88%。1H NMR：(400MHz，CDCl3)δ3.94-3.89(m，4H)，3.58-3.57(m，2H)，2.31-1.48(m，7H)。MS-ESI計算值[M+H]+ 159，實測值159。 Dissolve 1,4-dioxaspiro[4.4]nonane-7-carboxylic acid methyl ester (1.00g, 10.7mmol) in anhydrous tetrahydrofuran (30mL), under nitrogen protection, and slowly add lithium tetrahydroaluminum ( 531 mg, 13.9 mmol). The reaction solution was slowly raised to 25°C and stirred for 3 hours. Water (0.5 mL), 15% sodium hydroxide solution (0.5 mL), and water (1.5 mL) were sequentially added to the reaction solution. The insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain (1,4-dioxaspiro[4.4]nonane-7-yl)-methanol (1.5 mg, yellow oil), yield: 88%. 1 H NMR: (400 MHz, CDCl 3 ) δ 3.94-3.89 (m, 4H), 3.58-3.57 (m, 2H), 2.31-1.48 (m, 7H). MS-ESI calculated value [M+H] + 159, measured value 159.

第三步。 third step.

甲磺酸1，4-二氧雜螺[4.4]壬烷-7-基甲酯。 1,4-Dioxaspiro[4.4]nonane-7-yl methyl ester methanesulfonate.

將(1，4-二氧雜螺[4.4]壬烷-7-基)-甲醇(500mg，53.1mmol)和三乙胺(800mg，7.92mmol)溶於無水二氯甲烷(5mL)中，氮氣保護，0℃緩慢加入甲烷磺醯氯(433mg，3.80mmol)。反應液升至在25℃，攪拌2小時。加入水(40mL)淬滅反應，用乙酸乙酯萃取。合併有機相，依次用水(20mL x 2)，飽和氯化鈉溶液(50mL x 2)洗滌，用無水硫酸鎂乾燥，過濾，濾液減壓濃縮得到甲磺酸1，4-二氧雜螺[4.4]壬烷-7-基甲酯(800mg，黃色油狀)。MS-ESI計算值[M+H]+ 237，實測值237。 Dissolve (1,4-dioxaspiro[4.4]nonane-7-yl)-methanol (500mg, 53.1mmol) and triethylamine (800mg, 7.92mmol) in dry dichloromethane (5mL) under nitrogen Protect, slowly add methanesulfonyl chloride (433mg, 3.80mmol) at 0°C. The reaction solution was raised to 25°C and stirred for 2 hours. The reaction was quenched by adding water (40 mL) and extracted with ethyl acetate. The organic phases were combined, washed with water (20mL x 2), saturated sodium chloride solution (50mL x 2), dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 1,4-dioxaspiro methanesulfonate [4.4 ] Nonane-7-yl methyl ester (800 mg, yellow oil). MS-ESI calculated value [M+H] + 237, measured value 237.

第四步。 the fourth step.

(1，4-二氧雜螺[4.4]壬烷-7-基甲基)-3，7-二甲基1H-嘌呤-2，6(3H，7H)-二酮。 (1,4-dioxa-spiro [4.4] nonane-7-ylmethyl) -3,7-dimethyl-1 H - purine -2,6 (3 H, 7 H) - dione.

將甲磺酸1，4-二氧雜螺[4.4]壬烷-7-基甲酯(300mg，1.27mmol)溶於無水N，N-二甲基甲醯胺(10mL)中，氮氣保護，25℃加入碳酸鉀(350mg，2.54mmol)，碘化鉀(21.0mg，0.130mmol)，2，6-羥基-3，7-二甲基嘌呤(275mg，1.52mmol)。反應液加熱至在130℃，攪拌3小時。反應液降至25℃，加入水(40mL)淬滅反應，用乙酸乙酯(30mL x 2)萃取。合併有機相，用飽和氯化鈉溶液(100mL x 2)洗滌，用無水硫酸鎂乾 燥，過濾，濾液減壓濃縮得到(1，4-二氧雜螺[4.4]壬烷-7-基甲基)-3，7-二甲基1H-嘌呤-2，6(3H，7H)-二酮(200mg，白色固體)，產率：45%。MS-ESI計算值[M+H]+ 321，實測值321。 Dissolve 1,4-dioxaspiro[4.4]nonane-7- ylmethyl methanesulfonate (300mg, 1.27mmol) in anhydrous N , N -dimethylformamide (10mL) and protect with nitrogen. Potassium carbonate (350mg, 2.54mmol), potassium iodide (21.0mg, 0.130mmol), 2,6-hydroxy-3,7-dimethylpurine (275mg, 1.52mmol) were added at 25°C. The reaction solution was heated to 130°C and stirred for 3 hours. The reaction solution was reduced to 25°C, water (40 mL) was added to quench the reaction, and it was extracted with ethyl acetate (30 mL x 2). The organic phases were combined, washed with saturated sodium chloride solution (100mL x 2), dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain (1,4-dioxaspiro[4.4]nonane-7-ylmethyl )-3,7-Dimethyl 1 H -purine-2,6(3 H , 7 H )-dione (200 mg, white solid), yield: 45%. MS-ESI calculated value [M+H] + 321, measured value 321.

第五步。 the fifth step.

3，7-二甲基-1-(3-氧代-環戊基甲基)-1H-嘌呤-2，6(3H，7H)-二酮。 3,7-dimethyl-1- (3-oxo - cyclopentylmethyl) -1 H - purine -2,6 (3 H, 7 H) - dione.

將(1，4-二氧雜螺[4.4]壬烷-7-基甲基)-3，7-二甲基1H-嘌呤-2，6(3H，7H)-二酮(200mg，0.620mmol)溶於無水四氫呋喃(5mL)中，氮氣保護，25℃加入濃鹽酸(3mL)。反應液在25℃條件下攪拌1小時。加入水(60mL)稀釋，反應液用乙酸乙酯(20mL x 3)萃取。合併有機相，用飽和氯化鈉溶液(100mL x 2)洗滌，用無水硫酸鎂乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法純化(1：1石油醚/乙酸乙酯，Rf=0.3)，得到3，7-二甲基-1-(3-氧代-環戊基甲基)-1H-嘌呤-2，6(3H，7H)-二酮(100mg，黃色油狀)，產率：57%。MS-ESI計算值[M+H]+ 277，實測值277。 The (1,4-dioxaspiro[4.4]nonane-7-ylmethyl)-3,7-dimethyl 1 H -purine-2,6(3 H ,7 H )-dione (200mg , 0.620mmol) was dissolved in anhydrous tetrahydrofuran (5mL), protected by nitrogen, and concentrated hydrochloric acid (3mL) was added at 25°C. The reaction solution was stirred at 25°C for 1 hour. Water (60 mL) was added to dilute, and the reaction solution was extracted with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated sodium chloride solution (100mL x 2), dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified by silica gel column chromatography (1:1 petroleum ether/ethyl acetate, Rf=0.3 ) To obtain 3,7-dimethyl-1-(3-oxo-cyclopentylmethyl)-1 H -purine-2,6(3 H , 7 H )-dione (100 mg, yellow oil ), yield: 57%. MS-ESI calculated value [M+H] + 277, measured value 277.

第六步。 The sixth step.

1，3反式-1-((3-羥基-3-(三氟甲基)環戊基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1,3 trans-1-((3-hydroxy-3-(trifluoromethyl)cyclopentyl)methyl)-3,7-dimethyl-1 H -purine-2,6(3 H , 7 H )-Diketone.

1，3順式-1-((3-羥基-3-(三氟甲基)環戊基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1,3cis-1-((3-hydroxy-3-(trifluoromethyl)cyclopentyl)methyl)-3,7-dimethyl-1 H -purine-2,6(3 H , 7 H )-Diketone.

將3，7-二甲基-1-((3-氧代環戊基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮(100mg，0.362mmol)和氟化銫(11.0mg，0.0725mmol)溶於無水四氫呋喃(3mL)中，氮氣保護下加入三氟甲基三甲基矽烷(95.0mg，0.640mmol)。反應液緩慢加熱至30℃，攪拌12小時。向反應液中加入鹽酸水溶液(1N，5mL)，繼續在30℃攪拌0.5小時。向反應液中加入 水(50mL)稀釋，用飽和碳酸氫鈉水溶液(10mL)調pH值至7，用乙酸乙酯(30mL x 2)萃取。合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用製備高效液相色譜純化，得到兩個異構產物。 3,7-dimethyl-1 - ((3-oxo-cyclopentyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (100mg, 0.362mmol) and Cesium fluoride (11.0 mg, 0.0725 mmol) was dissolved in anhydrous tetrahydrofuran (3 mL), and trifluoromethyltrimethylsilane (95.0 mg, 0.640 mmol) was added under nitrogen protection. The reaction solution was slowly heated to 30°C and stirred for 12 hours. An aqueous hydrochloric acid solution (1N, 5 mL) was added to the reaction solution, and stirring was continued at 30°C for 0.5 hour. The reaction solution was diluted with water (50 mL), adjusted to pH 7 with saturated sodium bicarbonate aqueous solution (10 mL), and extracted with ethyl acetate (30 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified by preparative high performance liquid chromatography to obtain two isomerized products.

產物1(異構體1，第一個峰)(40.0mg，白色固體)，產率：32%。1H NMR：(400MHz，Methonal-d 4 )δ7.68(s，1H)，4.13-4.08(m，2H)，4.05(s，3H)，3.61(s，3H)，2.80-2.78(m，1H)，2.40-2.24(m，1H)，2.04-2.03(m，1H)，2.01-1.87(m，2H)，1.84-1.76(m，1H)，1.62-1.60(m，1H)。MS-ESI計算值[M+H]+ 347，實測值347。 Product 1 (isomer 1, first peak) (40.0 mg, white solid), yield: 32%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.68 (s, 1H), 4.13-4.08 (m, 2H), 4.05 (s, 3H), 3.61 (s, 3H), 2.80-2.78 (m, 1H), 2.40-2.24 (m, 1H), 2.04-2.03 (m, 1H), 2.01-1.87 (m, 2H), 1.84-1.76 (m, 1H), 1.62-1.60 (m, 1H). MS-ESI calculated value [M+H] + 347, measured value 347.

產物2(異構體2，第二個峰)(20.0mg，白色固體)，產率：16%。1H NMR：(400MHz，Methonal-d 4 )δ7.62(s，1H)，4.22-4.18(m，1H)，4.05-4.04(m，1H)，4.00(s，3H)，3.63(s，3H)，2.65-2.63(m，1H)，2.09-2.01(m，4H)，1.70-1.68(m，1H)，1.67-1.65(m，1H)。MS-ESI計算值[M+H]+ 347，實測值347。 Product 2 (isomer 2, second peak) (20.0 mg, white solid), yield: 16%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.62 (s, 1H), 4.22-4.18 (m, 1H), 4.05-4.04 (m, 1H), 4.00 (s, 3H), 3.63 (s, 3H), 2.65-2.63 (m, 1H), 2.09-2.01 (m, 4H), 1.70 to 1.68 (m, 1H), 1.67-1.65 (m, 1H). MS-ESI calculated value [M+H] + 347, measured value 347.

實施例11。 Example 11.

1-((4-羥基-4-(三氟甲基)環己基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 L - ((4-hydroxy-4- (trifluoromethyl) cyclohexyl) methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

Figure 105135042-A0305-02-0054-65
Figure 105135042-A0305-02-0054-65
Figure 105135042-A0305-02-0055-66
Figure 105135042-A0305-02-0055-66

第一步。 first step.

乙基1，4-二氧雜螺[4，5]癸烷-8-羧酸乙酯。 Ethyl 1,4-dioxaspiro[4,5]decane-8-carboxylic acid ethyl ester.

將乙基4-氧代環己烷羧酸(30.0g，176mmol)，乙二醇(22.0g，353mmol)和對甲苯磺酸(304mg，1.70mmol)溶於甲苯(315mL)中，加分水器後加熱回流反應過夜。反應液冷却至25℃，依次用水(300mL x 2)、飽和碳酸氫鈉(500mL x 2)洗滌，有機相用無水硫酸鎂乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法分離純化(1：1石油醚/乙酸乙酯，Rf=0.3)得到產物乙基1，4-二氧雜螺[4，5]癸烷-8-羧酸乙酯(37.2g，黃色液體)，產率：99%。MS-ESI計算值[M+H]+ 215，實測值215。 Dissolve ethyl 4-oxocyclohexanecarboxylic acid (30.0g, 176mmol), ethylene glycol (22.0g, 353mmol) and p-toluenesulfonic acid (304mg, 1.70mmol) in toluene (315mL), add water After the reactor, it was heated to reflux and reacted overnight. The reaction solution was cooled to 25°C, washed with water (300mL x 2) and saturated sodium bicarbonate (500mL x 2), the organic phase was dried with anhydrous magnesium sulfate, filtered, the filtrate was concentrated under reduced pressure, and then separated and purified by silica gel column chromatography (1 :1 petroleum ether/ethyl acetate, Rf=0.3) to obtain the product ethyl 1,4-dioxaspiro[4,5]decane-8-carboxylic acid ethyl ester (37.2g, yellow liquid), the yield: 99%. MS-ESI calculated value [M+H] + 215, measured value 215.

第二步。 The second step.

1，4-二氧雜螺[4，5]癸烷-8-基甲醇。 1,4-Dioxaspiro[4,5]decane-8-ylmethanol.

在氮氣保護，0℃時下將四氫鋁鋰(2.30g，61.0mmol)緩慢加入四氫呋喃(60mL)中，滴加乙基1，4-二氧雜螺[4，5]癸烷-8-羧酸乙酯(10.0g，42.0mmol)的四氫呋喃(40mL)溶液。反應緩慢升至25℃，攪拌3.5小時。反應液冷却至0℃，依次緩慢加入水(2.3g，127mmol)，15%氫氧化鈉(2.3g，8.60mmol)及水(6.9g，383mmol)。過濾，濾餅用四氫呋 喃(50mL x 3)洗滌，合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到產品1，4-二氧雜螺[4，5]癸烷-8-基甲醇(6.22g，黃色液體)，產率：89%。MS-ESI計算值[M+H]+ 173，實測值173。 Under nitrogen protection, at 0℃, slowly add lithium tetrahydroaluminum (2.30g, 61.0mmol) to tetrahydrofuran (60mL), add ethyl 1,4-dioxaspiro[4,5]decane-8- A solution of ethyl carboxylate (10.0 g, 42.0 mmol) in tetrahydrofuran (40 mL). The reaction was slowly raised to 25°C and stirred for 3.5 hours. The reaction solution was cooled to 0°C, and water (2.3g, 127mmol), 15% sodium hydroxide (2.3g, 8.60mmol) and water (6.9g, 383mmol) were slowly added in sequence. Filter, wash the filter cake with tetrahydrofuran (50mL x 3), combine the organic phases, dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain the product 1,4-dioxaspiro[4,5]decane-8-yl Methanol (6.22g, yellow liquid), yield: 89%. MS-ESI calculated value [M+H] + 173, measured value 173.

第三步。 third step.

1，4-二氧雜螺[4，5]癸烷-8-基甲基甲磺酸酯。 1,4-Dioxaspiro[4,5]decane-8-yl methanesulfonate.

將1，4-二氧雜螺[4，5]癸烷-8-基甲醇(2.00g，12.0mmol)及二異丙基乙基胺(3.10g，24.0mmol)溶於二氯甲烷(40mL)中，在0℃下緩慢加入甲烷磺醯氯(3.90g，30.0mmol)。反應液升至25℃，攪拌過夜。加入飽和氯化銨水溶液(100mL)淬滅反應，用乙酸乙酯萃取(200mL x 3)。合併有機相，用無水硫酸鎂乾燥，過濾，濾液減壓濃縮用矽膠柱色譜法分離純化(3：1石油醚/乙酸乙酯，Rf=0.4)，得到產物1，4-二氧雜螺[4，5]癸烷-8-基甲基甲磺酸酯(1.80g，黃色液體)，產率：60%。MS-ESI計算值[M+H]+ 251，實測值251。 1,4-Dioxaspiro[4,5]decane-8-ylmethanol (2.00g, 12.0mmol) and diisopropylethylamine (3.10g, 24.0mmol) were dissolved in dichloromethane (40mL ), slowly add methanesulfonyl chloride (3.90 g, 30.0 mmol) at 0°C. The reaction solution was raised to 25°C and stirred overnight. The reaction was quenched by adding saturated aqueous ammonium chloride solution (100 mL), and extracted with ethyl acetate (200 mL x 3). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (3:1 petroleum ether/ethyl acetate, Rf=0.4) to obtain the product 1,4-dioxaspiro [ 4,5] Decane-8-yl methanesulfonate (1.80 g, yellow liquid), yield: 60%. MS-ESI calculated value [M+H] + 251, measured value 251.

第四步。 the fourth step.

1-(1，4-二氧雜螺[4，5]癸烷-8-基甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1-(1,4-Dioxaspiro[4,5]decane-8-ylmethyl)-3,7-dimethyl-1 H -purine-2,6(3 H ,7 H )- Diketone.

將1，4-二氧雜螺[4，5]癸烷-8-基甲基甲磺酸酯(1.50g，6.00mmol)，3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(1.00g，6.00mmol)及碳酸鉀(2.50g，18.0mmol)，碘化鉀(100mg，0.600mmol)溶於N，N-二甲基甲醯胺(20mL)中，反應液加熱至130℃，攪拌3小時。反應液冷却至25℃，加入飽和食鹽水淬滅(100mL)反應，用乙酸乙酯萃取(500mL x 3)。合併有機相，用無水硫酸鎂乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法分離純化(1：1石油醚/乙酸乙酯，Rf=0.3)得到產物1-(1，4-二氧雜螺[4， 5]癸烷-8-基甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(1.75g，白色固體)，產率：63%。MS-ESI計算值[M+H]+ 335，實測值335。 1,4-Dioxaspiro[4,5]decane-8-yl methanesulfonate (1.50g, 6.00mmol), 3,7-dimethyl- 1H -purine-2,6 (3 H , 7 H )-dione (1.00g, 6.00mmol) and potassium carbonate (2.50g, 18.0mmol), potassium iodide (100mg, 0.600mmol) dissolved in N , N -dimethylformamide (20mL) In the middle, the reaction solution was heated to 130°C and stirred for 3 hours. The reaction solution was cooled to 25°C, and saturated brine was added to quench the reaction (100 mL), and extracted with ethyl acetate (500 mL x 3). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated under reduced pressure, and separated and purified by silica gel column chromatography (1:1 petroleum ether/ethyl acetate, Rf=0.3) to obtain the product 1-(1,4-dioxide) hetero-spiro [4, 5] decan-8-yl-methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (1.75 g of, as a white solid) , Yield: 63%. MS-ESI calculated value [M+H] + 335, measured value 335.

第五步。 the fifth step.

3，7-二甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮。 3,7-dimethyl-1 - ((4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione.

將1-(1，4-二氧雜螺[4，5]癸烷-8-基甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(1.50g，4.50mmol)溶於丙酮(15mL)中，加入鹽酸水溶液(2N，2.5mL)。反應25℃攪拌過夜，加入水(50mL)淬滅，用乙酸乙酯萃取(50mL x 3)。合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮用矽膠柱色譜法純化(1：3石油醚/乙酸乙酯，Rf=0.4)得到產物3，7-二甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮(1.02g，白色固體)，產率：78%。MS-ESI計算值[M+H]+ 291，實測值291。 1- (1,4-dioxaspiro [4,5] decan-8-yl-methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) -Dione (1.50 g, 4.50 mmol) was dissolved in acetone (15 mL), and aqueous hydrochloric acid (2N, 2.5 mL) was added. The reaction was stirred overnight at 25°C, quenched by adding water (50 mL), and extracted with ethyl acetate (50 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (1:3 petroleum ether/ethyl acetate, Rf=0.4) to obtain the product 3,7-dimethyl-1-( (4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (1.02 g of, as a white solid), yield: 78%. MS-ESI calculated value [M+H] + 291, measured value 291.

第六步。 The sixth step.

1-((4-羥基-4-(三氟甲基)環己基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 L - ((4-hydroxy-4- (trifluoromethyl) cyclohexyl) methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

將3，7-二甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮(100mg，0.330mmol)及氟化銫(60.0mg，0.350mmol)溶於四氫呋喃(5mL)中，在氮氣保護下緩慢加入三氟甲基三甲基矽烷(75.0mg，0.500mmol)。反應液在30℃下攪拌3小時。冷却至25℃，加入鹽酸水溶液(4N，3mL)，在25℃攪拌半小時，調節PH值至7，加水稀釋，用乙酸乙酯萃取(20mL x 3)。合併有機相，用無水硫酸鈉乾燥，過濾，減壓濃縮，用製備高效液相色譜純化得產物1-((4-羥基-4-(三氟甲基)環己 基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(24.0mg，白色固體)，產率：39%。 Combine 3,7-dimethyl-1-((4-oxocyclohexyl)methyl)-1 H -purine-2,6(3 H , 7 H )-dione (100mg, 0.330mmol) and fluoride Cesium (60.0 mg, 0.350 mmol) was dissolved in tetrahydrofuran (5 mL), and trifluoromethyltrimethylsilane (75.0 mg, 0.500 mmol) was slowly added under the protection of nitrogen. The reaction solution was stirred at 30°C for 3 hours. Cool to 25°C, add aqueous hydrochloric acid (4N, 3mL), stir at 25°C for half an hour, adjust the pH to 7, dilute with water, and extract with ethyl acetate (20mL x 3). The organic phases were combined, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by preparative high performance liquid chromatography to obtain the product 1-((4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3, 7-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (24.0 mg, white solid), yield: 39%.

1H NMR：(400MHz，Methonal-d 4 )δ7.86(s，1H)，4.04(d，J=7.2Hz，1H)，3.97(s，3H)，3.89(d，J=7.6Hz，1H)，3.53(s，3H)，2.06-1.97(m，2H)，1.88-1.77(m，3H)，1.62-143(m，4H)。MS-ESI計算值[M+H]+ 361，實測值361。 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.86 (s, 1H), 4.04 (d, J = 7.2 Hz, 1H), 3.97 (s, 3H), 3.89 (d, J = 7.6 Hz, 1H) ), 3.53 (s, 3H), 2.06-1.97 (m, 2H), 1.88-1.77 (m, 3H), 1.62-143 (m, 4H). MS-ESI calculated value [M+H] + 361, measured value 361.

實施例12。 Example 12.

Figure 105135042-A0305-02-0058-68
Figure 105135042-A0305-02-0058-68

第一步。 first step.

4-羥基-4-(三氟甲基)環己烷甲酸乙酯。 Ethyl 4-hydroxy-4-(trifluoromethyl)cyclohexanecarboxylate.

將4-氧代環己烷甲酸乙酯(10.0g，58.7mmol)溶於四氫呋喃(100mL)中，室溫條件下加入三氟甲基三甲基矽烷(12.5g，88.1mmol)和氟化銫(8.92g，58.7mmol)。反應液在室溫攪拌12小時，加入四丁基氟化銨(9.27g，29.4mmol)，室溫攪拌30分鐘後加入乙酸乙酯(80mL)稀釋，有機相用飽和碳酸氫鈉(50mL x 2)洗滌，無水硫酸鈉乾燥，過濾濃縮，用矽膠柱層析法分離純化(10：1石油醚/乙酸乙酯，Rf=0.5)得到4-羥基-4-(三氟甲基)環己烷甲酸乙酯(12.0g，無色油狀物)，產率：85%。1H NMR：(400MHz，Methanol-d 4 )δ4.20-4.12(m，2H)，2.03-1.86(m，9H)，1.29-1.25(m，3H)。 Dissolve ethyl 4-oxocyclohexanecarboxylate (10.0g, 58.7mmol) in tetrahydrofuran (100mL), add trifluoromethyltrimethylsilane (12.5g, 88.1mmol) and cesium fluoride at room temperature (8.92g, 58.7mmol). The reaction solution was stirred at room temperature for 12 hours, tetrabutylammonium fluoride (9.27g, 29.4mmol) was added, stirred at room temperature for 30 minutes and then diluted with ethyl acetate (80mL). The organic phase was diluted with saturated sodium bicarbonate (50mL x 2 ) Washed, dried with anhydrous sodium sulfate, filtered and concentrated, separated and purified by silica gel column chromatography (10:1 petroleum ether/ethyl acetate, Rf=0.5) to obtain 4-hydroxy-4-(trifluoromethyl)cyclohexane Ethyl formate (12.0 g, colorless oil), yield: 85%. 1 H NMR: (400MHz, Methanol- d 4 ) δ 4.20-4.12 (m, 2H), 2.03-1.86 (m, 9H), 1.29-1.25 (m, 3H).

MS-ESI計算值[M+H]+ 241，實測值241。 MS-ESI calculated value [M+H] + 241, measured value 241.

第二步。 The second step.

4-(羥基甲基)-1-(三氟甲基)環己醇。 4-(hydroxymethyl)-1-(trifluoromethyl)cyclohexanol.

將4-羥基-4-(三氟甲基)環己烷甲酸乙酯(12.00g，49.9mmol)溶於四氫呋喃(20mL)中，0℃下，加入四氫鋰鋁(3.79g，100mmol)，反應2小時。加入水(30mL)淬滅反應。用乙酸乙酯萃取(50mL x 3)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用用矽膠柱色譜法分離純化(1：1石油醚/乙酸乙酯，Rf=0.2)得到(4-(羥基甲基)-1-(三氟甲基)環己醇(9.00g，無色油狀物)，產率：91%。1H NMR：(400MHz，Methanol-d 4 )δ3.58-3.40(m，2H)，1.90-1.40(m，9H)。 Ethyl 4-hydroxy-4-(trifluoromethyl)cyclohexanecarboxylate (12.00g, 49.9mmol) was dissolved in tetrahydrofuran (20mL), and at 0°C, lithium aluminum tetrahydrogen (3.79g, 100mmol) was added, React for 2 hours. The reaction was quenched by adding water (30 mL). It was extracted with ethyl acetate (50mL x 3), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and separated and purified by silica gel column chromatography (1:1 petroleum ether/ethyl acetate, Rf=0.2) to obtain (4- (Hydroxymethyl)-1-(trifluoromethyl)cyclohexanol (9.00g, colorless oil), yield: 91%. 1 H NMR: (400MHz, Methanol- d 4 )δ3.58-3.40 (m, 2H), 1.90-1.40 (m, 9H).

第三步。 third step.

(4-羥基-4-(三氟甲基)環己基)甲基甲磺酸酯。 (4-Hydroxy-4-(trifluoromethyl)cyclohexyl)methanesulfonate.

將(4-(羥基甲基)-1-(三氟甲基)環己醇(11.0g，55.5mmol)和三乙胺(1.18g，11.6mmol)溶於二氯甲烷(80mL)中，0℃條件下加入甲磺醯氯(14.4g，125mmol)。反應液於室溫攪拌2小時後，加入二氯甲烷(60mL)稀釋，用飽和碳酸氫鈉(50mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱層析法分離純化(4：1石油醚/乙酸乙酯，Rf=0.5)得到(4-羥基-4-(三氟甲基)環己基)甲基甲磺酸酯(13.00g，無色油狀物)，產率：85%。1H NMR：(400MHz，Methanol-d 4 )δ4.25-4.01(m，2H)，3.10-3.07(m，3H)，2.03-1.24(m，9H)。 (4-(hydroxymethyl)-1-(trifluoromethyl)cyclohexanol (11.0g, 55.5mmol) and triethylamine (1.18g, 11.6mmol) were dissolved in dichloromethane (80mL), 0 Add methanesulfonyl chloride (14.4g, 125mmol) at ℃. After stirring the reaction solution at room temperature for 2 hours, add dichloromethane (60mL) to dilute, wash with saturated sodium bicarbonate (50mL x 2), and dry with anhydrous sodium sulfate , Filter, concentrate the filtrate under reduced pressure, and use silica gel column chromatography to separate and purify (4:1 petroleum ether/ethyl acetate, Rf=0.5) to obtain (4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl Methanesulfonate (13.00g, colorless oil), yield: 85%. 1 H NMR: (400MHz, Methanol- d 4 ) δ 4.25-4.01 (m, 2H), 3.10-3.07 (m, 3H) ), 2.03-1.24 (m, 9H).

第四步。 the fourth step.

1-(((1S，4S)-4-羥基-4-(三氟甲基)環己基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1-(((1 S ,4 S )-4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3,7-dimethyl-1 H -purine-2,6(3 H , 7 H )-Diketone.

將(4-羥基-4-(三氟甲基)環己基)甲基甲磺酸酯(10.0g，36.2mmol)溶於N，N-二甲基甲醯胺(100mL)中，反應液於室溫條件下加入3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(6.52g，36.2mmol)，碳酸鉀(7.50g，54.3mmol)和碘化鉀(184mg，1.11mmol)。反應液加熱至100℃，反應5小時，反應液濃縮，加入乙酸乙酯(100mL)稀釋，有機相用飽和碳酸氫鈉(50mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，通過製備型SFC來分離，分離條件：色譜柱：Chiralpak AD-3 150 x 4.6mm I.D.，3um流動相：乙醇(0.05%二乙胺)in CO2 from 5% to 40% at 2.5mL/min波長：220nm得到產物1(2.5g，白色固體)(異構體1，第1個峰)，產率：19%。1H NMR：(400MHz，Methanol-d 4 )δ7.88(s，1H)，4.02(d，J=7.6Hz，2H)，3.98(s，3H)，3.53(s，3H)，2.16-2.02(m，1H)，1.99-1.98(m，2H)，1.87-1.80(m，2H)，1.60-1.49(m，2H)，1.48-1.46(m，2H)。MS-ESI計算值[M+H]+ 361，實測值361。 (4-Hydroxy-4-(trifluoromethyl)cyclohexyl)methanesulfonate (10.0g, 36.2mmol) was dissolved in N , N -dimethylformamide (100mL), and the reaction solution was 3,7-dimethyl -1 H was added at room temperature - purin -2,6 (3 H, 7 H) - dione (6.52g, 36.2mmol), potassium carbonate (7.50g, 54.3mmol) and potassium iodide (184mg, 1.11mmol). The reaction solution was heated to 100°C and reacted for 5 hours. The reaction solution was concentrated and diluted with ethyl acetate (100mL). The organic phase was washed with saturated sodium bicarbonate (50mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Separation by preparative SFC, separation conditions: Column: Chiralpak AD-3 150 x 4.6mm ID, 3um mobile phase: ethanol (0.05% diethylamine) in CO 2 from 5% to 40% at 2.5mL/min wavelength : 220nm to obtain product 1 (2.5g, white solid) (isomer 1, first peak), yield: 19%. 1 H NMR: (400MHz, Methanol- d 4 )δ7.88(s, 1H), 4.02(d, J =7.6Hz, 2H), 3.98(s, 3H), 3.53(s, 3H), 2.16-2.02 (m, 1H), 1.99-1.98 (m, 2H), 1.87-1.80 (m, 2H), 1.60-1.49 (m, 2H), 1.48-1.46 (m, 2H). MS-ESI calculated value [M+H] + 361, measured value 361.

產物2(2.40g，白色固體)(異構體2，第二個峰)，產率：19%。1H NMR：(400MHz，CDCl3)δ7.88(s，1H)，3.99(s，3H)，3.90(d，J=7.6Hz，2H)，3.54(s，3H)，1.84-1.81(m，3H)，1.58-1.46(m，6H)。MS-ESI計算值[M+H]+ 361，實測值361。 Product 2 (2.40 g, white solid) (isomer 2, second peak), yield: 19%. 1 H NMR: (400MHz, CDCl 3 ) δ 7.88 (s, 1H), 3.99 (s, 3H), 3.90 (d, J = 7.6 Hz, 2H), 3.54 (s, 3H), 1.84-1.81 (m , 3H), 1.58-1.46 (m, 6H). MS-ESI calculated value [M+H] + 361, measured value 361.

實施例13。 Example 13.

1-((4-羥基-4-甲基環己基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 L - ((4-hydroxy-cyclohexyl) methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

Figure 105135042-A0305-02-0060-70
Figure 105135042-A0305-02-0060-70
Figure 105135042-A0305-02-0061-71
Figure 105135042-A0305-02-0061-71

將3，7-二甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮(50.0mg，0.170mmol)溶於四氫呋喃(2mL)中。氮氣保護，在-78℃緩慢加入甲基格氏試劑(3M乙醚溶劑，0.4mL，1.20mmol)。反應液在-78℃攪拌0.5小時，緩慢升至0℃繼續攪拌0.5小時。加入氯化銨飽和溶液淬滅，調節pH值至7。用乙酸乙酯萃取，無水硫酸鈉乾燥，過濾。濾液減壓濃縮用製備高效液相色譜純化得產物1-((4-羥基-4-甲基環己基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(8.0mg，白色固體)，產率：16%。1H NMR：(400MHz，Methonal-d 4 )δ7.86(s，1H)，3.97(s，3H)，3.88(d，J=7.6Hz，2H)，3.52(s，3H)，1.85-1.78(m，1H)，1.73-1.57(m，3H)，1.46-1.33(m，2H)，1.32-1.15(m，6H)。MS-ESI計算值[M+H-H2O]+ 289，實測值289。 3,7-dimethyl-1 - ((4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (50.0mg, 0.170mmol) was dissolved in Tetrahydrofuran (2mL). Under nitrogen protection, slowly add methyl Grignard reagent (3M ether solvent, 0.4 mL, 1.20 mmol) at -78°C. The reaction solution was stirred at -78°C for 0.5 hour, then slowly raised to 0°C and stirring continued for 0.5 hour. Add saturated ammonium chloride solution to quench, adjust the pH value to 7. It was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by preparative high performance liquid chromatography to obtain the product 1-((4-hydroxy-4-methylcyclohexyl)methyl)-3,7-dimethyl-1 H -purine-2,6(3 H , 7 H )-diketone (8.0 mg, white solid), yield: 16%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.86 (s, 1H), 3.97 (s, 3H), 3.88 (d, J = 7.6 Hz, 2H), 3.52 (s, 3H), 1.85-1.78 (m, 1H), 1.73-1.57 (m, 3H), 1.46-1.33 (m, 2H), 1.32-1.15 (m, 6H). MS-ESI calculated value [M+HH 2 O] + 289, measured value 289.

實施例14。 Example 14.

1-((4-乙基-4-羥基環己基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 L - ((4-ethyl-4-hydroxy-cyclohexyl) methyl) -1H- purine-3,7-dimethyl -2,6 (3 H, 7 H) - dione.

Figure 105135042-A0305-02-0061-74
Figure 105135042-A0305-02-0061-74

將3，7-二甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮(50.0mg，0.170mmol)溶於四氫呋喃(2mL)中。氮氣保護，在-78℃緩慢加入乙基格氏試劑(3M在乙醚溶劑，0.4mL，1.20mmol)。 反應液在-78℃攪拌0.5小時，緩慢升至0℃繼續攪拌0.5小時。加入氯化銨飽和溶液淬滅，調節pH值至7。用乙酸乙酯萃取，無水硫酸鈉乾燥，過濾。濾液減壓濃縮用製備高效液相色譜純化得產物1-((4-乙基-4-羥基環己基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(42.0mg，白色固體)，產率：77%。1H NMR：(400MHz，Methonal-d 4 )δ7.86(s，1H)，397(s，3H)，3.88(d，J=7.6Hz，2H)，3.54-3.50(m，3H)，1.93-180(m，1H)，1.76-1.72(m，2H)，1.66-151(m，3H)，1.38-1.28(m，3H)，1.27-1.13(m，2H)，0.89(t，J=7.2Hz，3H)。MS-ESI計算值[M+H-H2O]+ 303，實測值303。 3,7-dimethyl-1 - ((4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (50.0mg, 0.170mmol) was dissolved in Tetrahydrofuran (2mL). Under nitrogen protection, slowly add ethyl Grignard reagent (3M in ether solvent, 0.4 mL, 1.20 mmol) at -78°C. The reaction solution was stirred at -78°C for 0.5 hour, then slowly raised to 0°C and stirring continued for 0.5 hour. Add saturated ammonium chloride solution to quench, adjust the pH value to 7. It was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by preparative high performance liquid chromatography to obtain the product 1-((4-ethyl-4-hydroxycyclohexyl)methyl)-3,7-dimethyl-1H-purine-2,6(3 H , 7 H )-diketone (42.0 mg, white solid), yield: 77%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.86 (s, 1H), 397 (s, 3H), 3.88 (d, J = 7.6 Hz, 2H), 3.54-3.50 (m, 3H), 1.93 -180 (m, 1H), 1.76-1.72 (m, 2H), 1.66-151 (m, 3H), 1.38-1.28 (m, 3H), 1.27-1.13 (m, 2H), 0.89 (t, J = 7.2Hz, 3H). MS-ESI calculated value [M+HH 2 O] + 303, measured value 303.

實施例15。 Example 15.

Figure 105135042-A0305-02-0062-75
Figure 105135042-A0305-02-0062-75

第一步。 first step.

乙基2-(1，4-二氧雜螺[4.5]癸烷-8-亞基)乙酸甲酯。 Ethyl methyl 2-(1,4-dioxaspiro[4.5]decane-8-ylidene) acetate.

將膦醯基乙酸三乙酯(12.2g，54.4mmol)溶於四氫呋喃(100mL)，0℃下分批加入氫化鈉(1.92g，48.0mmol)，氮氣保護下攪拌反應30分鐘。0℃下將溶於四氫呋喃(15mL)的1，4-環己二酮單乙二醇縮酮(5.00g，32.0mmol)溶液滴加到反應液中，反應液於25℃下攪拌反應3小時。加入水(25mL)淬滅反應，用二氯甲烷(20mL x 3)萃取。合併有機相，飽和食鹽水洗滌(20mL)，無水硫酸鈉乾燥，減壓濃縮，剩餘物剩餘物用矽膠柱色譜法純化(5：1石油醚/乙酸乙酯，Rf=0.3)，得乙基2-(1，4-二氧雜螺[4.5]癸烷-8-亞基)乙酸甲酯(6.30g，無色油狀物)，產率：93%。1H NMR：(400MHz，CDCl3)δ5.67(s，1H)，4.15(q，J=7.2Hz，2H)，3.98(s，4H)，3.00(t，J=6.4Hz，2H)，2.38(t，J=6.4Hz，2H)，1.84-1.68(m，4H)，1.28(t，J=7.2Hz，3H)。MS-ESI計算值[M+H]+ 227，實測值227。 Triethyl phosphinoacetate (12.2 g, 54.4 mmol) was dissolved in tetrahydrofuran (100 mL), sodium hydride (1.92 g, 48.0 mmol) was added in portions at 0° C., and the reaction was stirred for 30 minutes under nitrogen protection. A solution of 1,4-cyclohexanedione monoethylene ketal (5.00 g, 32.0 mmol) dissolved in tetrahydrofuran (15 mL) was added dropwise to the reaction solution at 0°C, and the reaction solution was stirred and reacted at 25°C for 3 hours . The reaction was quenched by adding water (25 mL) and extracted with dichloromethane (20 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (5:1 petroleum ether/ethyl acetate, Rf=0.3) to obtain ethyl Methyl 2-(1,4-dioxaspiro[4.5]decane-8-ylidene)acetate (6.30g, colorless oil), yield: 93%. 1 H NMR: (400MHz, CDCl 3 ) δ 5.67 (s, 1H), 4.15 (q, J = 7.2 Hz, 2H), 3.98 (s, 4H), 3.00 (t, J = 6.4 Hz, 2H), 2.38 (t, J = 6.4 Hz, 2H), 1.84-1.68 (m, 4H), 1.28 (t, J = 7.2 Hz, 3H). MS-ESI calculated value [M+H] + 227, measured value 227.

第二步。 The second step.

乙基2-(1，4-二氧雜螺[4.5]癸烷-8-基)乙酸乙酯。 Ethyl ethyl 2-(1,4-dioxaspiro[4.5]decane-8-yl)acetate.

將甲基2-(1，4-二氧雜螺[4.5]癸烷-8-亞基)乙酸甲酯(3.80g，17.9mmol)溶於甲醇(50mL)，加入幹鈀碳(鈀10%，水1%，400mg)，室溫下，反應液於氫氣(50psi)下反應18小時。反應液過濾，濾液減壓濃縮得到甲基2-(1，4-二氧雜螺[4.5]癸烷-8-基)乙酸乙酯(3.50g，無色油狀物)，產率：91%。 Dissolve methyl 2-(1,4-dioxaspiro[4.5]decane-8-ylidene) acetate (3.80g, 17.9mmol) in methanol (50mL), add dry palladium on carbon (palladium 10% , Water 1%, 400mg), at room temperature, the reaction solution was reacted under hydrogen (50psi) for 18 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain ethyl 2-(1,4-dioxaspiro[4.5]decane-8-yl)ethyl acetate (3.50g, colorless oil), yield: 91% .

1H NMR：(400MHz，CDCl3)δ4.12(q，J=7.2Hz，2H)，3.93(s，4H)，2.22(d，J=7.2Hz，2H)，1.90-1.64(m，5H)，1.63-1.48(m，2H)，1.40-1.16(m，5H)。MS-ESI計算值[M+H]+ 229，實測值229。 1 H NMR: (400MHz, CDCl 3 ) δ 4.12 (q, J = 7.2 Hz, 2H), 3.93 (s, 4H), 2.22 (d, J = 7.2 Hz, 2H), 1.90 to 1.64 (m, 5H) ), 1.63-1.48 (m, 2H), 1.40-1.16 (m, 5H). MS-ESI calculated value [M+H] + 229, measured value 229.

第三步。 third step.

2-(1，4-二氧雜螺[4.5]癸烷-8-基)乙醇。 2-(1,4-Dioxaspiro[4.5]decane-8-yl)ethanol.

將乙基2-(1，4-二氧雜螺[4.5]癸烷-8-基)乙酸乙酯(1.00g，4.38mmol)溶於四氫呋喃(20mL)，0℃下分批加入四氫鋰鋁(216mg，5.69mmol)，氮氣保護下攪拌反應18小時。反應液冷却至0℃，依次緩慢加入水(0.2mL)，15%氫氧化鈉水溶液(0.2mL)及水(0.6mL)。過濾，濾液減壓濃縮得到產物2-(1，4-二氧雜螺[4.5]癸烷-8-基)乙醇(780mg，黃色油狀物)，產率：96%。 Ethyl 2-(1,4-dioxaspiro[4.5]decane-8-yl)ethyl acetate (1.00g, 4.38mmol) was dissolved in tetrahydrofuran (20mL), and tetrahydrolithium was added in portions at 0°C Aluminum (216 mg, 5.69 mmol) was stirred for 18 hours under nitrogen protection. The reaction solution was cooled to 0°C, and water (0.2 mL), 15% aqueous sodium hydroxide solution (0.2 mL) and water (0.6 mL) were slowly added in sequence. After filtration, the filtrate was concentrated under reduced pressure to obtain the product 2-(1,4-dioxaspiro[4.5]decane-8-yl)ethanol (780 mg, yellow oil), yield: 96%.

1H NMR：(400MHz，CDCl3)δ3.94(s，4H)，3.69(t，J=6.4Hz，2H)，1.79-1.65(m，4H)，1.59-1.38(m，5H)，1.34-1.17(m，2H)。MS-ESI計算值[M+H]+ 187，實測值187。 1 H NMR: (400MHz, CDCl 3 ) δ 3.94 (s, 4H), 3.69 (t, J = 6.4 Hz, 2H), 1.79-1.65 (m, 4H), 1.59-1.38 (m, 5H), 1.34 -1.17 (m, 2H). MS-ESI calculated value [M+H] + 187, measured value 187.

第四步。 the fourth step.

2-(1，4-二氧雜螺[4.5]癸烷-8-基)乙基甲磺酸酯 2-(1,4-dioxaspiro[4.5]decane-8-yl)ethyl methanesulfonate

將2-(1，4-二氧雜螺[4.5]癸烷-8-基)乙醇(400mg，2.15mmol)及三乙胺(435mg，4.30mmol)溶於二氯甲烷(10mL)中，在0℃下緩慢加入甲烷磺醯氯(369mg，3.23mmol)。反應液於0℃下，攪拌4小時。加水(10mL)淬滅反應，用二氯甲烷萃取(30mL x 2)。合併有機相，用飽和碳酸氫鈉水溶液(50mL)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到2-(1，4-二氧雜螺[4.5]癸烷-8-基)乙基甲磺酸酯(500mg粗品，黃色油狀物)。 Dissolve 2-(1,4-dioxaspiro[4.5]decane-8-yl)ethanol (400mg, 2.15mmol) and triethylamine (435mg, 4.30mmol) in dichloromethane (10mL). Slowly add methanesulfonyl chloride (369mg, 3.23mmol) at 0°C. The reaction solution was stirred at 0°C for 4 hours. The reaction was quenched by adding water (10 mL) and extracted with dichloromethane (30 mL x 2). The organic phases were combined, washed with saturated aqueous sodium bicarbonate solution (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2-(1,4-dioxaspiro[4.5]decane-8-yl)ethane Mesylate (500 mg crude, yellow oil).

1H NMR：(400MHz，CDCl3)δ4.28(t，J=6.4Hz，2H)，3.94(s，4H)，3.01(s，3H)，1.76-1.63(m，6H)，1.60-1.43(m，3H)，1.37-1.21(m，2H)。MS-ESI計算值[M+H]+ 265，實測值265。 1 H NMR: (400MHz, CDCl 3 ) δ 4.28 (t, J = 6.4 Hz, 2H), 3.94 (s, 4H), 3.01 (s, 3H), 1.76-1.63 (m, 6H), 1.60-1.43 (m, 3H), 1.37-1.21 (m, 2H). MS-ESI calculated value [M+H] + 265, measured value 265.

第五步。 the fifth step.

1-(2-(1，4-二氧雜螺[4.5]癸烷-8-基)乙基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-(2-(1,4-Dioxaspiro[4.5]decane-8-yl)ethyl)-3,7-dimethyl-1 H -purine-2,6-(3 H ,7 H )-Diketone.

將3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(204mg，1.13mmol)溶於N，N-二甲基甲醯胺(15mL)，加入2-(1，4-二氧雜螺[4.5]癸烷-8-基)乙基甲磺酸酯(300mg，1.13mmol)，碳酸鉀(312mg，2.26mmol)和碘化鉀(225mg，1.36mmol)。反應液加熱到120℃，攪拌3小時。減壓濃縮，剩餘物用矽膠柱色譜法純化(1：1石油醚/乙酸乙酯，Rf=0.2)，得1-[2-(1，4-二氧雜螺[4.5]癸烷-8-基)乙基]-3，7-二甲基嘌呤-2，6-二酮(190mg，白色固體)，產率：48%。 The 3,7-dimethyl -1 H - purine -2,6- (3 H, 7 H) - dione (204mg, 1.13mmol) was dissolved in N, N - dimethylformamide (15mL), Add 2-(1,4-dioxaspiro[4.5]decane-8-yl)ethyl methanesulfonate (300mg, 1.13mmol), potassium carbonate (312mg, 2.26mmol) and potassium iodide (225mg, 1.36mmol) ). The reaction solution was heated to 120°C and stirred for 3 hours. Concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (1:1 petroleum ether/ethyl acetate, Rf=0.2) to obtain 1-[2-(1,4-dioxaspiro[4.5]decane-8 -Yl)ethyl]-3,7-dimethylpurine-2,6-dione (190 mg, white solid), yield: 48%.

1H NMR：(400MHz，CDCl3)δ7.50(s，1H)，4.09-4.03(m，2H)，4.02(s，3H)，3.99(s，4H)，3.57(s，1H)，1.90-1.70(m，5H)，1.68-1.47(m，6H)，1.45-1.31(m，2H)。MS-ESI計算值[M+H]+ 349，實測值349。 1 H NMR: (400MHz, CDCl 3 ) δ 7.50 (s, 1H), 4.09-4.03 (m, 2H), 4.02 (s, 3H), 3.99 (s, 4H), 3.57 (s, 1H), 1.90 -1.70 (m, 5H), 1.68-1.47 (m, 6H), 1.45-1.31 (m, 2H). MS-ESI calculated value [M+H] + 349, measured value 349.

第六步。 The sixth step.

3，7-二甲基-1-(2-(4-氧代環己基)乙基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3,7-dimethyl-1- (2- (4-oxo-cyclohexyl) ethyl) -1 H - purine -2,6- (3 H, 7 H) - dione.

將1-[2-(1，4-二氧雜螺[4.5]癸烷-8-基)乙基]-3，7-二甲基嘌呤-2，6-二酮(190mg，545umol)溶於四氫呋喃(3mL)，加入濃鹽酸(1mL)。反應液室溫下攪拌18小時。反應液減壓濃縮，水相用飽和碳酸氫鈉中和到pH至7，用乙酸乙酯(20mL x 2)萃取，合併有機相，用飽和食鹽水(10mL)洗滌，無水硫酸鈉乾燥，過濾，減壓濃縮，剩餘物用矽膠柱色譜法純化(乙酸乙酯，Rf=0.3)，得3，7-二甲基-1-(2-(4-氧代環己基)乙基)-1H-嘌呤-2，6-(3H，7H)-二酮(150mg，無色油狀物)，產率：90%。 Dissolve 1-[2-(1,4-dioxaspiro[4.5]decane-8-yl)ethyl]-3,7-dimethylpurine-2,6-dione (190mg, 545umol) To tetrahydrofuran (3 mL), concentrated hydrochloric acid (1 mL) was added. The reaction solution was stirred at room temperature for 18 hours. The reaction solution was concentrated under reduced pressure, the aqueous phase was neutralized to pH 7 with saturated sodium bicarbonate, extracted with ethyl acetate (20 mL x 2), the organic phases were combined, washed with saturated brine (10 mL), dried with anhydrous sodium sulfate, and filtered , Concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate, Rf=0.3) to obtain 3,7-dimethyl-1-(2-(4-oxocyclohexyl)ethyl)-1 H -purine-2,6-( 3H , 7H )-dione (150mg, colorless oil), yield: 90%.

MS-ESI計算值[M+H]+ 305，實測值305。 MS-ESI calculated value [M+H] + 305, measured value 305.

第七步。 The seventh step.

3，7-二甲基-1-(2-(4-(三氟甲基)-4-((三甲基甲矽烷基)氧基)環己基)乙基)-1-嘌呤-2，6-(3H，7H)-二酮。 3,7-Dimethyl-1-(2-(4-(trifluoromethyl)-4-((trimethylsilyl)oxy)cyclohexyl)ethyl)-1-purine-2, 6-(3H,7H)-dione.

將3，7-二甲基-1-[2-(4-氧環己基)乙基]嘌呤-2，6-二酮(145mg，0.476mmol)及氟化銫(7.2mg，0.0476mmol)溶於四氫呋喃(10mL)中，在氮氣保護下緩慢加入三氟甲基三甲基矽烷(203mg，1.43mmol)。反應液在25℃下攪拌18小時。加入水(20mL)稀釋反應液，用乙酸乙酯(15mL x 2)萃取，合併有機相，飽和食鹽水洗滌(10mL)，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得3，7-二甲基-1-(2-(4-(三氟甲基)-4-((三甲基甲矽烷基)氧基)環己基)乙基)-1-嘌呤-2，6-(3H，7H)-二酮(170mg，無色液體)，產率：80%。 Dissolve 3,7-dimethyl-1-[2-(4-oxocyclohexyl)ethyl]purine-2,6-dione (145mg, 0.476mmol) and cesium fluoride (7.2mg, 0.0476mmol) In tetrahydrofuran (10 mL), trifluoromethyltrimethylsilane (203 mg, 1.43 mmol) was slowly added under the protection of nitrogen. The reaction solution was stirred at 25°C for 18 hours. Add water (20mL) to dilute the reaction solution, extract with ethyl acetate (15mL x 2), combine the organic phases, wash with saturated brine (10mL), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain 3,7- Dimethyl-1-(2-(4-(trifluoromethyl)-4-((trimethylsilyl)oxy)cyclohexyl)ethyl)-1-purine-2,6-(3 H , 7 H )-diketone (170 mg, colorless liquid), yield: 80%.

MS-ESI計算值[M+H]+ 447，實測值447。 MS-ESI calculated value [M+H] + 447, measured value 447.

第八步。 The eighth step.

將3，7-二甲基-1-[2-[4-(三氟甲基)-4-三甲基矽氧基-環己基]乙基]嘌呤-2，6-二酮(160mg，0.358mmol)溶於四氫呋喃(3mL)，加入濃鹽酸(12M，0.107mL)。反應液於25℃攪拌18小時。加水稀釋，飽和碳酸氫鈉溶液(10mL)調節pH至7，用乙酸乙酯萃取(10mL x 2)。合併有機相，用無水硫酸鈉乾燥，過濾，減壓濃縮，用製備高效液相色譜純化得產物1(40.0mg，白色固體)(異構體1，第一個峰)，產率：27%。1H NMR：(400MHz，CDCl3)δ8.01(s，1H)，4.09-3.94(m，5H)，3.53(s，3H)，1.97-1.79(m，4H)，1.76-1.62(m，3H)，1.61-1.45(m，4H)。MS-ESI計算值[M+H]+ 375，實測值375。和產物2(15.0mg，白色固體)(異構體2，第二個峰)，產率：10%。1H NMR：(400MHz，CDCl3)δ8.01(s，1H)，4.09-3.95(m，5H)，3.53(s，3H)，1.87-1.68(m，4H)，1.64-1.48 (m，4H)，1.46-1.25(m，3H)。MS-ESI計算值[M+H]+ 375，實測值375。 3,7-Dimethyl-1-[2-[4-(trifluoromethyl)-4-trimethylsiloxy-cyclohexyl]ethyl]purine-2,6-dione (160mg, 0.358 mmol) was dissolved in tetrahydrofuran (3 mL), and concentrated hydrochloric acid (12M, 0.107 mL) was added. The reaction solution was stirred at 25°C for 18 hours. Dilute with water, adjust the pH to 7 with saturated sodium bicarbonate solution (10 mL), and extract with ethyl acetate (10 mL x 2). The organic phases were combined, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by preparative high performance liquid chromatography to obtain product 1 (40.0 mg, white solid) (isomer 1, first peak), yield: 27% . 1 H NMR: (400MHz, CDCl 3 ) δ8.01 (s, 1H), 4.09-3.94 (m, 5H), 3.53 (s, 3H), 1.97-1.79 (m, 4H), 1.76-1.62 (m, 3H), 1.61-1.45 (m, 4H). MS-ESI calculated value [M+H] + 375, measured value 375. And product 2 (15.0 mg, white solid) (isomer 2, second peak), yield: 10%. 1 H NMR: (400MHz, CDCl 3 ) δ 8.01 (s, 1H), 4.09-3.95 (m, 5H), 3.53 (s, 3H), 1.87-1.68 (m, 4H), 1.64-1.48 (m, 4H), 1.46-1.25 (m, 3H). MS-ESI calculated value [M+H]+ 375, measured value 375.

實施例16。 Example 16.

1-((4-羥基-1-甲基-4-(三氟甲基)環己基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7-dimethyl-1H-purine-2,6-(3 H , 7 H )-Diketone.

Figure 105135042-A0305-02-0067-76
Figure 105135042-A0305-02-0067-76

第一步。 first step.

乙基-8-甲基-1，4-二氧雜螺[4.5]癸烷-8-甲酸三級丁酯 Ethyl-8-methyl-1,4-dioxaspiro[4.5]decane-8-carboxylic acid tertiary butyl ester

將乙基1，4-二氧雜螺[4.5]癸烷-8-羧酸乙酯(5.00g，23.3mmol)溶於無水四氫呋喃(100mL)中，在氮氣保護，-78℃時緩慢滴加二異丙基氨基鋰溶液(2M四氫呋喃溶液，14.0mL，28.0mmol)，反應液在-78℃攪拌1小時。緩慢加入碘甲烷(6.62g，46.7mmol)，繼續攪拌1小時。加入水(100mL)淬滅反應。反應液用乙酸乙酯(100mL x 3)萃取，合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法純化(10：1石油醚/乙酸乙酯，Rf=0.4)得到乙基-8-甲基-1，4-二氧雜螺[4.5] 癸烷-8-甲酸三級丁酯(5.00g，黃色油狀物)，產率：94%。1H NMR：(400MHz，Methonal-d 4 )δ4.16-4.10(m，2H)，3.93-3.86(m，4H)，2.13-2.06(m，2H)，1.61-1.48(m，6H)，1.25-1.22(m，3H)，1.15(s，3H)。 Ethyl 1,4-dioxaspiro[4.5]decane-8-ethyl carboxylate (5.00g, 23.3mmol) was dissolved in anhydrous tetrahydrofuran (100mL), and slowly added dropwise at -78℃ under nitrogen protection Lithium diisopropylamide solution (2M tetrahydrofuran solution, 14.0 mL, 28.0 mmol), the reaction solution was stirred at -78°C for 1 hour. Slowly add methyl iodide (6.62 g, 46.7 mmol) and continue stirring for 1 hour. The reaction was quenched by adding water (100 mL). The reaction solution was extracted with ethyl acetate (100mL x 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified by silica gel column chromatography (10:1 petroleum ether/ethyl acetate, Rf=0.4 ) Obtain ethyl-8-methyl-1,4-dioxaspiro[4.5]decane-8-carboxylic acid tertiary butyl ester (5.00 g, yellow oil), yield: 94%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 4.16-4.10 (m, 2H), 3.93-3.86 (m, 4H), 2.13-2.06 (m, 2H), 1.61-1.48 (m, 6H), 1.25-1.22 (m, 3H), 1.15 (s, 3H).

第二步。 The second step.

乙基-1-甲基-4-氧代環己烷羧酸。 Ethyl-1-methyl-4-oxocyclohexanecarboxylic acid.

將乙基-8-甲基-1，4-二氧雜螺[4.5]癸烷-8-甲酸三級丁酯(5.00g，21.9mmol)溶於四氫呋喃(50mL)，0℃下滴加1N鹽酸水溶液(20mL)後於20℃攪拌1小時。混合物冷却到0℃，加入碳酸氫鈉溶液(50mL)淬滅反應。混合物用乙酸乙酯(100mL x 3)萃取。有機相用飽和食鹽水(100mL x 3)洗滌，無水硫酸鈉乾燥後減壓濃縮。用矽膠柱色譜法純化(10：1石油醚/乙酸乙酯，Rf=0.4)得到乙基-1-甲基-4-氧代環己烷羧酸(3.00g，無色油狀物)，產率：74%。1H NMR：(400MHz，Methonal-d 4 )δ4.26-4.11(m，2H)，2.46-2.29(m，5H)，1.74-1.55(m，3H)，1.33-1.26(m，6H)。 Dissolve ethyl-8-methyl-1,4-dioxaspiro[4.5]decane-8-carboxylic acid tertiary butyl ester (5.00g, 21.9mmol) in tetrahydrofuran (50mL), add 1N dropwise at 0℃ The aqueous hydrochloric acid solution (20 mL) was then stirred at 20°C for 1 hour. The mixture was cooled to 0°C, and sodium bicarbonate solution (50 mL) was added to quench the reaction. The mixture was extracted with ethyl acetate (100 mL x 3). The organic phase was washed with saturated brine (100 mL x 3), dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purified by silica gel column chromatography (10:1 petroleum ether/ethyl acetate, Rf=0.4) to obtain ethyl-1-methyl-4-oxocyclohexanecarboxylic acid (3.00g, colorless oil) as Rate: 74%. 1 H NMR: (400 MHz, Methonal- d 4 ) δ 4.26-4.11 (m, 2H), 2.46-2.29 (m, 5H), 1.74-1.55 (m, 3H), 1.33-1.26 (m, 6H).

第三步。 third step.

乙基4-羥基-1-甲基-4-(三氟甲基)環己烷羧酸。 Ethyl 4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexanecarboxylic acid.

將乙基-1-甲基-4-氧代環己烷羧酸(3.00g，16.3mmol)，氟化銫(247mg，1.63mmol)溶於四氫呋喃(50mL)中，然後0℃加入三甲基矽三氟甲基(4.63g，35.3mmol)。反應液在20℃氮氣保護下反應6小時。然後加入4N鹽酸水溶液(4mL)。混合物在室溫氮氣保護下反應6小時。加入碳酸氫鈉飽和溶液(30mL)淬滅反應，用乙酸乙酯萃取(100mL x 3)，有機相用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法純化(10：1石油醚/乙酸乙酯，Rf=0.3)得到乙基4-羥基-1-甲基-4-(三氟甲基)環己 烷羧酸(3.00g，無色油狀物)，產率：73%。1H NMR：(400MHz，Methanol-d 4 )δ4.20-4.12(m，2H)，2.03-1.31(m，8H)，1.29-1.23(m，6H)。 Dissolve ethyl-1-methyl-4-oxocyclohexanecarboxylic acid (3.00g, 16.3mmol) and cesium fluoride (247mg, 1.63mmol) in tetrahydrofuran (50mL), then add trimethyl at 0°C Sitrifluoromethyl (4.63 g, 35.3 mmol). The reaction solution was reacted at 20°C under nitrogen protection for 6 hours. Then 4N aqueous hydrochloric acid solution (4 mL) was added. The mixture was reacted for 6 hours under nitrogen protection at room temperature. The reaction was quenched by adding saturated sodium bicarbonate solution (30mL), extracted with ethyl acetate (100mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified by silica gel column chromatography (10:1 petroleum Ether/ethyl acetate, Rf=0.3) to obtain ethyl 4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexanecarboxylic acid (3.00g, colorless oil), yield: 73% . 1 H NMR: (400MHz, Methanol- d 4 ) δ 4.20-4.12 (m, 2H), 2.03-1.31 (m, 8H), 1.29-1.23 (m, 6H).

第四步。 the fourth step.

4-(羥甲基)-4-甲基-1-(三氟甲基)環己醇。 4-(hydroxymethyl)-4-methyl-1-(trifluoromethyl)cyclohexanol.

將乙基4-羥基-1-甲基-4-(三氟甲基)環己烷羧酸(3.00g，11.8mmol)溶於無水四氫呋喃(50mL)中，0℃下加入四氫鋁鋰(896mg，23.6mmol)。反應液升溫至25℃，攪拌1小時。加水(20mL)淬滅，用乙酸乙酯(50mL x 3)萃取，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法純化(3：1石油醚/乙酸乙酯，Rf=0.2)，得到4-(羥甲基)-4-甲基-1-(三氟甲基)環己醇(2.00g，無色油狀物)，產率：80%。1H NMR：(400MHz，Methanol-d 4 )δ3.25(s，2H)，1.76-1.64(m，6H)，1.29-1.26(m，2H),0.93-0.91(m，3H)。 Ethyl 4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexanecarboxylic acid (3.00g, 11.8mmol) was dissolved in anhydrous tetrahydrofuran (50mL), and lithium tetrahydroaluminum ( 896mg, 23.6mmol). The reaction solution was heated to 25°C and stirred for 1 hour. Quench with water (20mL), extract with ethyl acetate (50mL x 3), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify with silica gel column chromatography (3:1 petroleum ether/ethyl acetate, Rf=0.2 ) To obtain 4-(hydroxymethyl)-4-methyl-1-(trifluoromethyl)cyclohexanol (2.00 g, colorless oil), yield: 80%. 1 H NMR: (400MHz, Methanol- d 4 ) δ 3.25 (s, 2H), 1.76-1.64 (m, 6H), 1.29-1.26 (m, 2H), 0.93-0.91 (m, 3H).

第五步。 the fifth step.

(4-羥基-1-甲基-4-(三氟甲基)環己基)甲基甲磺酸酯。將4-(羥甲基)-4-甲基-1-(三氟甲基)環己醇(2.00g，9.42mmol)溶解在二氯甲烷(30mL)中，在0℃下加入三乙胺(953mg，9.42mmol)和甲烷磺醯氯(1.08g，9.42mmol)。反應液在0℃下反應2小時。加入碳酸氫鈉飽和水溶液(10mL)淬滅，用二氯甲烷(50mL x 3)萃取，合併有機相，用飽和氯化鈉溶液(50mL x 3)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到(4-羥基-1-甲基-4-(三氟甲基)環己基)甲基甲磺酸酯(2.00g，黃色油狀物)，產率：73%。 (4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methanesulfonate. Dissolve 4-(hydroxymethyl)-4-methyl-1-(trifluoromethyl)cyclohexanol (2.00g, 9.42mmol) in dichloromethane (30mL), add triethylamine at 0°C (953mg, 9.42mmol) and methanesulfonyl chloride (1.08g, 9.42mmol). The reaction solution was reacted at 0°C for 2 hours. It was quenched by adding saturated aqueous sodium bicarbonate solution (10 mL), extracted with dichloromethane (50 mL x 3), combined the organic phases, washed with saturated sodium chloride solution (50 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was reduced in pressure Concentrated to obtain (4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methanesulfonate (2.00 g, yellow oil), yield: 73%.

第六步。 The sixth step.

1-((4-羥基-1-甲基-4-(三氟甲基)環己基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7-dimethyl-1 H -purine-2,6-(3 H ,7 H )-Diketone.

(4-羥基-1-甲基-4-(三氟甲基)環己基)甲基甲磺酸酯(100mg，0.344mmol)，3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(62.1mg，0.344mmol)，碘化鉀(5.70mg，0.0344mmol)和碳酸鉀(47.6mg，0.344mmol)溶於無水N，N-二甲基甲醯胺(5mL)中。反應液微波加熱至150℃，反應4小時。反應液冷却至20℃，過濾，用製備高效液相色譜純化，得到1-((4-羥基-1-甲基-4-(三氟甲基)環己基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(3.0mg，白色固體)，產率：2%。1H NMR：(400MHz，Methonal-d 4 )δ7.88(s，1H)，3.98(s，3H)，3.96(s，2H)，3.54(s，3H)，1.81-1.64(m，6H)，1.63-1.34(m，2H)，1.00(s，3H)。MS-ESI計算值[M+H]+ 375，實測值375。 (4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methanesulfonate (100mg, 0.344mmol), 3,7-dimethyl- 1H -purine-2,6 -(3 H , 7 H )-dione (62.1 mg, 0.344 mmol), potassium iodide (5.70 mg, 0.0344 mmol) and potassium carbonate (47.6 mg, 0.344 mmol) are dissolved in anhydrous N , N -dimethylformamide (5mL). The reaction solution was heated to 150° C. in a microwave and reacted for 4 hours. The reaction solution was cooled to 20°C, filtered, and purified by preparative high performance liquid chromatography to obtain 1-((4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7- Dimethyl- 1H -purine-2,6-( 3H , 7H )-dione (3.0mg, white solid), yield: 2%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.88 (s, 1H), 3.98 (s, 3H), 3.96 (s, 2H), 3.54 (s, 3H), 1.81-1.64 (m, 6H) , 1.63-1.34 (m, 2H), 1.00 (s, 3H). MS-ESI calculated value [M+H] + 375, measured value 375.

實施例17。 Example 17.

1-((4-羥基-1-甲基-4-(三氟甲基)環己基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7-dimethyl-1 H -purine-2,6-(3 H ,7 H )-Diketone.

Figure 105135042-A0305-02-0070-77
Figure 105135042-A0305-02-0070-77
Figure 105135042-A0305-02-0071-78
Figure 105135042-A0305-02-0071-78

第一步。 first step.

乙基8-(甲氧基甲基)-1，4-二氧雜螺[4.5]癸烷-8-羧酸乙酯。 Ethyl 8-(methoxymethyl)-1,4-dioxaspiro[4.5]decane-8-carboxylic acid ethyl ester.

將乙基1，4-二氧雜螺[4.5]癸烷-8-羧酸乙酯(5.00g，23.3mmol)溶於無水四氫呋喃(100mL)中，在氮氣保護，-78℃時緩慢滴加二異丙基氨基鋰溶液(2M正己烷溶液，14.0mL，28.0mmol)，反應液在-78℃攪拌1小時。緩慢加入甲氧基溴甲烷(5.83g，46.7mmol)，繼續攪拌1小時。加入水(100mL)淬滅反應。反應液用乙酸乙酯(100mL x 3)萃取，合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法純化(10：1石油醚/乙酸乙酯，Rf=0.3)得到乙基8-(甲氧基甲基)-1，4-二氧雜螺[4.5]癸烷-8-羧酸乙酯(5.00g，黃色油狀物)，產率：83%。1H NMR：(400MHz，Methanol-d 4 )δ4.18(q，J=6.8Hz，2H)，3.94(s，4H)，3.55(s，2H),3.33(s，3H)，2.14-2.12(m，2H)，1.65-1.57(m，6H),1.26(t，J=6.8Hz，3H)。 Ethyl 1,4-dioxaspiro[4.5]decane-8-ethyl carboxylate (5.00g, 23.3mmol) was dissolved in anhydrous tetrahydrofuran (100mL), and slowly added dropwise at -78℃ under nitrogen protection Lithium diisopropylamide solution (2M n-hexane solution, 14.0 mL, 28.0 mmol), the reaction solution was stirred at -78°C for 1 hour. Slowly add methoxy bromomethane (5.83 g, 46.7 mmol) and continue stirring for 1 hour. The reaction was quenched by adding water (100 mL). The reaction solution was extracted with ethyl acetate (100mL x 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified by silica gel column chromatography (10:1 petroleum ether/ethyl acetate, Rf=0.3 ) Obtain ethyl 8-(methoxymethyl)-1,4-dioxaspiro[4.5]decane-8-carboxylic acid ethyl ester (5.00 g, yellow oil), yield: 83%. 1 H NMR: (400MHz, Methanol- d 4 )δ4.18(q, J =6.8Hz, 2H), 3.94(s, 4H), 3.55(s, 2H), 3.33(s, 3H), 2.14-2.12 (m, 2H), 1.65-1.57 (m, 6H), 1.26 (t, J = 6.8 Hz, 3H).

第二步。 The second step.

乙基-1-(甲氧基甲基)-4-氧代環己烷羧酸乙酯。 Ethyl-1-(methoxymethyl)-4-oxocyclohexanecarboxylic acid ethyl ester.

將乙基8-(甲氧基甲基)-1，4-二氧雜螺[4.5]癸烷-8-羧酸乙酯(5.00g，19.4mmol)溶於四氫呋喃(50mL)，0℃下滴加1N稀鹽酸(10mL)後於20℃攪拌1小時。混合物冷却到0℃，加入碳酸氫鈉溶液(50mL)淬滅反應。混合物用乙酸乙酯(100mL x 3)萃取。有機相用飽和食鹽水(100mL x 3)洗滌，無水硫酸鈉乾燥後減壓濃縮。用矽膠柱色譜法純化(10：1石油醚/乙酸乙酯，Rf=0.4)得到乙基-1-(甲氧基甲基)-4-氧代環己烷羧酸乙酯(3.00g，白色油狀物)，產率：73%。1H NMR：(400MHz，Methanol-d 4 )δ4.25(q，J=6.8Hz，2H)，3.52(s，2H)，3.34(s，3H)，2.52-2.30(m，6H)，1.82-1.78(m，2H),1.30(t，J=6.8Hz，3H)。 Ethyl 8-(methoxymethyl)-1,4-dioxaspiro[4.5]decane-8-carboxylic acid ethyl ester (5.00g, 19.4mmol) was dissolved in tetrahydrofuran (50mL) at 0°C 1N diluted hydrochloric acid (10 mL) was added dropwise and stirred at 20°C for 1 hour. The mixture was cooled to 0°C, and sodium bicarbonate solution (50 mL) was added to quench the reaction. The mixture was extracted with ethyl acetate (100 mL x 3). The organic phase was washed with saturated brine (100 mL x 3), dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purified by silica gel column chromatography (10:1 petroleum ether/ethyl acetate, Rf=0.4) to obtain ethyl-1-(methoxymethyl)-4-oxocyclohexanecarboxylic acid ethyl ester (3.00g, White oil), yield: 73%. 1 H NMR: (400MHz, Methanol- d 4 ) δ 4.25 (q, J = 6.8 Hz, 2H), 3.52 (s, 2H), 3.34 (s, 3H), 2.52-2.30 (m, 6H), 1.82 -1.78 (m, 2H), 1.30 (t, J = 6.8 Hz, 3H).

第三步。 third step.

4-羥基-1-(甲氧基甲基)-4-(三氟甲基)環己烷羧酸乙酯。 Ethyl 4-hydroxy-1-(methoxymethyl)-4-(trifluoromethyl)cyclohexanecarboxylate.

將乙基-1-(甲氧基甲基)-4-氧代環己烷羧酸乙酯(3.00g，14.0mmol)，氟化銫(243mg，1.40mmol)溶於四氫呋喃(50mL)中，然後0℃加入三甲基矽三氟甲基(3.98g，28.0mmol)。反應液在20℃氮氣保護下反應6小時。然後加入4N稀鹽酸(7mL)。混合物在室溫氮氣保護下反應6小時。加入碳酸氫鈉飽和溶液(30mL)淬滅反應，用乙酸乙酯萃取(100mL x 3)，有機相用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法純化(10：1石油醚/乙酸乙酯，Rf=0.4)得到4-羥基-1-(甲氧基甲基)-4-(三氟甲基)環己烷羧酸乙酯(1.7g，無色油狀物)，產率：43%。1H NMR：(400MHz，Methonal-d 4 )4.18-4.09(m，2H)，3.61(s，2H)，3.33(s，3H)，1.84-1.71(m，8H)，1.28-1.25(m，3H)。 Ethyl-1-(methoxymethyl)-4-oxocyclohexanecarboxylate (3.00g, 14.0mmol), cesium fluoride (243mg, 1.40mmol) were dissolved in tetrahydrofuran (50mL), Then trimethylsilyltrifluoromethyl (3.98g, 28.0mmol) was added at 0°C. The reaction solution was reacted at 20°C under nitrogen protection for 6 hours. Then 4N dilute hydrochloric acid (7 mL) was added. The mixture was reacted for 6 hours under nitrogen protection at room temperature. The reaction was quenched by adding saturated sodium bicarbonate solution (30mL), extracted with ethyl acetate (100mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified by silica gel column chromatography (10:1 petroleum Ether/ethyl acetate, Rf=0.4) to give ethyl 4-hydroxy-1-(methoxymethyl)-4-(trifluoromethyl)cyclohexanecarboxylate (1.7g, colorless oil), Yield: 43%. 1 H NMR: (400MHz, Methonal- d 4 ) 4.18-4.09 (m, 2H), 3.61 (s, 2H), 3.33 (s, 3H), 1.84-1.71 (m, 8H), 1.28-1.25 (m, 3H).

第四步。 the fourth step.

4-(羥基甲基)-4-(甲氧基甲基)-1-(三氟甲基)環己醇。 將4-羥基-1-(甲氧基甲基)-4-(三氟甲基)環己烷羧酸乙酯(1.50g，5.28mmol)溶於無水四氫呋喃(50mL)中，0℃下加入四氫鋁鋰(220mg，5.81mmol)。反應液升溫至25℃，攪拌1小時。加水(20mL)淬滅，用乙酸乙酯(50mL x 3)萃取，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法純化(1：1石油醚/乙酸乙酯，Rf=0.2)，得到4-(羥基甲基)-4-(甲氧基甲基)-1-(三氟甲基)環己醇(1.20g，無色油狀物)，產率：84%。1H NMR：(400MHz，Methanol-d 4 )δ3.33-3.32(m，7H)，1.67-1.63(m，4H)，1.52-1.48(m，4H)。 4-(hydroxymethyl)-4-(methoxymethyl)-1-(trifluoromethyl)cyclohexanol. Ethyl 4-hydroxy-1-(methoxymethyl)-4-(trifluoromethyl)cyclohexanecarboxylate (1.50g, 5.28mmol) was dissolved in dry tetrahydrofuran (50mL) and added at 0°C Lithium aluminum tetrahydrogen (220 mg, 5.81 mmol). The reaction solution was heated to 25°C and stirred for 1 hour. Quench with water (20mL), extract with ethyl acetate (50mL x 3), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify with silica gel column chromatography (1:1 petroleum ether/ethyl acetate, Rf=0.2 ) To obtain 4-(hydroxymethyl)-4-(methoxymethyl)-1-(trifluoromethyl)cyclohexanol (1.20 g, colorless oil), yield: 84%. 1 H NMR: (400MHz, Methanol- d 4 ) δ3.33-3.32 (m, 7H), 1.67-1.63 (m, 4H), 1.52-1.48 (m, 4H).

第五步。 the fifth step.

(4-羥基-1-甲基-4-(三氟甲基)環己基)甲基甲磺酸酯。 (4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methanesulfonate.

將4-(羥基甲基)-4-(甲氧基甲基)-1-(三氟甲基)環己醇(1.20g，4.95mmol)溶解在二氯甲烷(20mL)中，在0℃下加入三乙胺(851mg，9.91mmol)和甲烷磺醯氯(851mg，7.43mmol)。反應液在0℃下反應2小時。加入碳酸氫鈉飽和水溶液(10mL)淬滅，用二氯甲烷(50mL x 3)萃取，合併有機相，用飽和氯化鈉溶液(50mL x 3)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到(4-羥基-1-甲基-4-(三氟甲基)環己基)甲基甲磺酸酯(1.30g，黃色油狀物)，產率：92%。 Dissolve 4-(hydroxymethyl)-4-(methoxymethyl)-1-(trifluoromethyl)cyclohexanol (1.20g, 4.95mmol) in dichloromethane (20mL) at 0°C Add triethylamine (851mg, 9.91mmol) and methanesulfonyl chloride (851mg, 7.43mmol). The reaction solution was reacted at 0°C for 2 hours. It was quenched by adding saturated aqueous sodium bicarbonate solution (10 mL), extracted with dichloromethane (50 mL x 3), combined the organic phases, washed with saturated sodium chloride solution (50 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was reduced in pressure Concentrated to obtain (4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methanesulfonate (1.30 g, yellow oil), yield: 92%.

第六步。 The sixth step.

1-((4-羥基-1-甲基-4-(三氟甲基)環己基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7-dimethyl-1 H -purine-2,6-(3 H ,7 H )-Diketone.

(4-羥基-1-甲基-4-(三氟甲基)環己基)甲基甲磺酸酯(300mg，1.05mmol)，3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(189mg，1.05mmol)，碘化鉀(17.4mg，0.105mmol)和碳酸鉀(435mg，3.15mmol)溶於無水N，N-二甲基甲醯胺(5mL)中。反應液微波加熱至150℃，反應 2小時。反應液冷却至20℃，過濾，用製備高效液相色譜純化，得到1-((4-羥基-1-甲基-4-(三氟甲基)環己基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(12.0mg，白色固體)，產率：3%。1H NMR：(400MHz，Methonal-d 4 )δ7.87(s，1H)，4.06(s，2H)，3.83(s，3H)，3.98(s，3H)，3.53(s，2H)，3.42(s，3H)，1.69-1.58(m，8H)。MS-ESI計算值[M+H]+ 405，實測值405。 (4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methanesulfonate (300mg, 1.05mmol), 3,7-dimethyl- 1H -purine-2,6 -(3 H , 7 H )-dione (189mg, 1.05mmol), potassium iodide (17.4mg, 0.105mmol) and potassium carbonate (435mg, 3.15mmol) dissolved in anhydrous N , N -dimethylformamide (5mL )in. The reaction solution was heated to 150°C in microwave and reacted for 2 hours. The reaction solution was cooled to 20°C, filtered, and purified by preparative high performance liquid chromatography to obtain 1-((4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7- dimethyl -1 H - purine -2,6- (3 H, 7 H) - dione (12.0 mg, white solid), yield: 3%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.87 (s, 1H), 4.06 (s, 2H), 3.83 (s, 3H), 3.98 (s, 3H), 3.53 (s, 2H), 3.42 (s, 3H), 1.69-1.58 (m, 8H). MS-ESI calculated value [M+H] + 405, measured value 405.

實施例18。 Example 18.

1-((4-(3-羥基戊3-基)-環己基)甲基)-3，7-二甲基1H-嘌呤-2，6(3H，7H)-二酮。 1-((4-(3-hydroxypent3-yl)-cyclohexyl)methyl)-3,7-dimethyl 1 H -purine-2,6(3 H ,7 H )-dione.

Figure 105135042-A0305-02-0074-79
Figure 105135042-A0305-02-0074-79

第一步。 first step.

4-羥甲基環己烷羧酸甲酯。 Methyl 4-hydroxymethylcyclohexanecarboxylate.

將1，4-環己烷甲酸甲酯(1.20g，6.45mmol)溶於無水四氫呋喃(20mL)中，氮氣保護，0℃下時緩慢滴加硼烷二甲硫醚(10M，1.0mL，10.3mmol)，反應液在0℃攪拌0.5小時，緩慢升至25℃，繼續攪拌1小時。加入水(40mL)淬滅反應，反應液用乙酸乙酯萃取。合併有機相，依次用水，飽和氯化鈉溶液洗滌，用無水硫酸鎂乾燥，過濾，濾液減壓濃縮得到 4-羥甲基環己烷羧酸甲酯(1.00g，白色固體)，產率：91%。1H NMR：(400MHz，CDCl3)δ3.67(s，3H)，3.48-3.46(m，2H)，2.26-2.25(m，1H)，2.05-2.01(m，2H)，1.89-1.85(m，2H)，1.47-1.43(m，2H)，1.31(s，1H)，1.01-0.98(m，2H)。MS-ESI計算值[M+H]+ 173，實測值173。 Methyl 1,4-cyclohexanecarboxylate (1.20g, 6.45mmol) was dissolved in anhydrous tetrahydrofuran (20mL), protected by nitrogen, and borane dimethyl sulfide (10M, 1.0mL, 10.3) was slowly added dropwise at 0°C. mmol), the reaction solution was stirred at 0°C for 0.5 hour, slowly raised to 25°C, and stirring continued for 1 hour. Water (40 mL) was added to quench the reaction, and the reaction solution was extracted with ethyl acetate. The organic phases were combined, washed with water, saturated sodium chloride solution, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain methyl 4-hydroxymethylcyclohexanecarboxylate (1.00 g, white solid). The yield: 91%. 1 H NMR: (400MHz, CDCl 3 ) δ 3.67 (s, 3H), 3.48-3.46 (m, 2H), 2.26-2.25 (m, 1H), 2.05-2.01 (m, 2H), 1.89-1.85 ( m, 2H), 1.47-1.43 (m, 2H), 1.31 (s, 1H), 1.01-0.98 (m, 2H). MS-ESI calculated value [M+H] + 173, measured value 173.

第二步。 The second step.

4-甲磺醯氧基甲基-環己烷羧酸甲酯。 4-Methanesulfonyloxymethyl-cyclohexanecarboxylic acid methyl ester.

將4-羥甲基環己烷羧酸甲酯(900mg，5.20mmol)和三乙胺(1.58g，15.6mmol)溶於無水二氯甲烷(5mL)中，氮氣保護，0℃下加入甲烷磺醯氯(720mg，6.30mmol)。反應液升至25℃，攪拌2小時。加入水(60mL)淬滅反應，反應液用乙酸乙酯萃取。合併有機相，依次用水，飽和氯化鈉溶液洗滌，用無水硫酸鎂乾燥，過濾，濾液減壓濃縮，用製備TLC板純化(3：1石油醚/乙酸乙酯，Rf=0.5)，得產物4-甲磺醯氧基甲基-環己烷羧酸甲酯(1.00g，白色固體)，產率：91%。1H NMR：(400MHz，CDCl3)δ3.67(s，3H)，3.48-3.46(m，2H)，3.01(s，3H)，2.26-2.25(m，1H)，2.05-2.01(m，2H)，1.89-1.85(m，2H)，1.47-1.43(m，2H)，1.31(s，1H)，1.01-0.98(m，2H)。MS-ESI計算值[M+H]+ 251，實測值251。 Methyl 4-hydroxymethylcyclohexanecarboxylate (900mg, 5.20mmol) and triethylamine (1.58g, 15.6mmol) were dissolved in dry dichloromethane (5mL), protected by nitrogen, and methanesulfonate was added at 0°C Chlorine (720 mg, 6.30 mmol). The reaction solution was raised to 25°C and stirred for 2 hours. Water (60 mL) was added to quench the reaction, and the reaction solution was extracted with ethyl acetate. The organic phases were combined, washed with water, saturated sodium chloride solution, dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified with a preparative TLC plate (3:1 petroleum ether/ethyl acetate, Rf=0.5) to obtain the product Methyl 4-methanesulfonyloxymethyl-cyclohexanecarboxylate (1.00 g, white solid), yield: 91%. 1 H NMR: (400MHz, CDCl 3 ) δ 3.67 (s, 3H), 3.48-3.46 (m, 2H), 3.01 (s, 3H), 2.26-2.25 (m, 1H), 2.05-2.01 (m, 2H), 1.89-1.85 (m, 2H), 1.47-1.43 (m, 2H), 1.31 (s, 1H), 1.01-0.98 (m, 2H). MS-ESI calculated value [M+H] + 251, measured value 251.

第三步。 third step.

4-((3，7-二甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)-環己烷羧酸甲酯。 4-((3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)methyl)-cyclohexanecarboxylic acid methyl ester.

將4-甲磺醯氧基甲基-環己烷羧酸甲酯(580mg，2.32mmol)溶於5mL無水N，N-二甲基甲醯胺中，在氮氣保護下25℃加入碳酸鉀(640mg，4.64mmol)，碘化鉀(38.0mg，0.230mmol)，2，6-羥基-3，7-二甲基嘌 呤(501mg，2.80mmol)。反應液在130℃下攪拌3小時。40mL水加入反應液用乙酸乙酯萃取，合併有機相，依次用水、飽和氯化鈉溶液洗滌，用無水硫酸鎂乾燥，過濾，濾液減壓濃縮，用高效製備板純化得到產物甲基4-((3，7-二甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)-環己烷羧酸甲酯(400mg，白色固體)，產率：52%。MS-ESI計算值[M+H]+ 335，實測值335。 4-Methanesulfonyloxymethyl-cyclohexanecarboxylic acid methyl ester (580mg, 2.32mmol) was dissolved in 5mL of anhydrous N , N -dimethylformamide, and potassium carbonate ( 640 mg, 4.64 mmol), potassium iodide (38.0 mg, 0.230 mmol), 2,6-hydroxy-3,7-dimethylpurine (501 mg, 2.80 mmol). The reaction solution was stirred at 130°C for 3 hours. 40mL of water was added to the reaction solution and extracted with ethyl acetate. The organic phases were combined, washed successively with water and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified with a high-efficiency preparation plate to obtain the product methyl 4-( (3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)methyl)-cyclohexanecarboxylic acid methyl ester (400mg , White solid), yield: 52%. MS-ESI calculated value [M+H] + 335, measured value 335.

第四步。 the fourth step.

1-((4-(3-羥基戊3-基)-環己基)甲基)-3，7-二甲基1H-嘌呤-2，6(3H，7H)-二酮。 1-((4-(3-hydroxypent3-yl)-cyclohexyl)methyl)-3,7-dimethyl 1 H -purine-2,6(3 H ,7 H )-dione.

將甲基4-((3，7-二甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)-環己烷羧酸(100mg，0.30mmol)溶於5mL無水四氫呋喃中，在氮氣保護下-65℃時緩慢滴加乙基溴化鎂溶液(3M乙醚溶液，1mL，3.00mmol)，反應液在-65℃下攪拌2小時。反應液加入水(40mL)，用乙酸乙酯萃取，合併有機相，用飽和氯化鈉溶液(50mL)洗滌，用無水硫酸鎂乾燥，過濾，濾液減壓濃縮，用製備高效液相色譜純化得到產物1-((4-(3-羥基戊3-基)-環己基)甲基)-3，7-二甲基1H-嘌呤-2，6(3H，7H)-二酮(20.0mg，白色固體)，產率：19%。1H NMR：(400MHz，CDCl3)δ7.52(s，1H)，4.00(s，3H)，3.90-3.88(m，2H)，3.59(s，3H)，1.80-1.74(m，6H)，1.50-1.45(m，4H)，1.11-1.10(m，4H)，0.86-0.82(m，6H)。MS ESI計算值[M+H]+ 363，實測值363。 The methyl 4-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)-cyclohexanecarboxylic acid ( 100mg, 0.30mmol) was dissolved in 5mL of anhydrous tetrahydrofuran, and ethylmagnesium bromide solution (3M ether solution, 1mL, 3.00mmol) was slowly added dropwise at -65°C under nitrogen protection, and the reaction solution was stirred at -65°C for 2 hours The reaction solution was added with water (40mL), extracted with ethyl acetate, combined the organic phases, washed with saturated sodium chloride solution (50mL), dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified by preparative high performance liquid chromatography The product 1-((4-(3-hydroxypent3-yl)-cyclohexyl)methyl)-3,7-dimethyl 1 H -purine-2,6(3 H ,7 H )-dione (20.0 mg, white solid), yield: 19%. 1 H NMR: (400MHz, CDCl 3 ) δ 7.52 (s, 1H), 4.00 (s, 3H), 3.90-3.88 (m, 2H), 3.59 (s, 3H), 1.80-1.74 (m, 6H), 1.50-1.45 (m, 4H), 1.11-1.10 (m, 4H), 0.86-0.82 (m, 6H). MS ESI calculated value [M+H ] + 363, the measured value is 363.

實施例19。 Example 19.

3，7-二甲基-1-[[反式-4-(2，2，2-三氟-1-羥基-1-甲基-乙基)環己基]甲基]嘌呤-2，6-二酮。 3,7-Dimethyl-1-[[trans-4-(2,2,2-trifluoro-1-hydroxy-1-methyl-ethyl)cyclohexyl]methyl]purine-2,6 -Diketone.

Figure 105135042-A0305-02-0077-80
Figure 105135042-A0305-02-0077-80

第一步。 first step.

反式-4-羥甲基環己烷羧酸甲酯。 Trans-4-hydroxymethylcyclohexanecarboxylic acid methyl ester.

將反式環己烷-1，4-二羧酸單甲酯(5.00g，26.8mmol)溶於四氫呋喃(100mL)中，0℃下加入硼烷二甲硫醚(3.06g，40.3mmol)，室溫反應2小時。加入飽和甲醇(50mL)淬滅反應。濃縮後加水(50mL)用乙酸乙酯萃取(10mL x 3)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到反式-4-羥甲基環己烷羧酸甲酯(4.00g，黃色油狀)，產率：87%。1H NMR：(400MHz，Methonal-d 4)δ3.67(s，3H)，3.43-3.38(m，2H)，2.31-2.54(m，1H)，2.03-1.98(m，2H)，1.90-1.82(m，2H)，1.45-1.38(m，3H)，1.03-0.99(m，2H)。MS-ESI計算值[M+H]+ 173，實測值173。 Dissolve trans-cyclohexane-1,4-dicarboxylic acid monomethyl ester (5.00 g, 26.8 mmol) in tetrahydrofuran (100 mL), add borane dimethyl sulfide (3.06 g, 40.3 mmol) at 0°C, React at room temperature for 2 hours. The reaction was quenched by adding saturated methanol (50 mL). After concentration, water (50mL) was added and extracted with ethyl acetate (10mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain methyl trans-4-hydroxymethylcyclohexanecarboxylate (4.00g, yellow oil) State), yield: 87%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 3.67 (s, 3H), 3.43-3.38 (m, 2H), 2.31-2.54 (m, 1H), 2.03-1.98 (m, 2H), 1.90- 1.82 (m, 2H), 1.45-1.38 (m, 3H), 1.03-0.99 (m, 2H). MS-ESI calculated value [M+H] + 173, measured value 173.

第二步。 The second step.

反式-4-甲磺醯氧基甲基-環己烷羧酸甲酯。 Trans-4-Methanesulfonyloxymethyl-cyclohexanecarboxylic acid methyl ester.

將反式-4-羥甲基環己烷羧酸甲酯(4.00g，23.2mmol)和三乙胺(7.05g，69.6mmol)溶於二氯甲烷(50mL)中，0℃下加入甲烷磺醯氯(7.98g，69.6mmol)。反應液緩慢升至室溫，攪拌2小時。加入碳酸氫鈉水溶液(50mL)淬滅反應。用二氯甲烷萃取(20mL x 3)。合併有機相，用飽和食鹽水(30mL)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到反式-4-甲磺醯氧基甲基-環己烷羧酸甲酯(5.80g，黃色油狀)，產率：99%。1H NMR：(400MHz，Methonal-d 4)δ4.10-4.03(m，2H)，3.65(s，3H)，3.07(s，3H)，2.42-2.31(m，1H)，2.10-2.03(m，2H)，1.90-1.82(m，2H)，1.75-1.66(m，1H)，1.48-1.42(m，2H)，1.21-1.10(m，2H)。MS-ESI計算值[M+H]+ 251，實測值251。 Dissolve methyl trans-4-hydroxymethylcyclohexanecarboxylate (4.00g, 23.2mmol) and triethylamine (7.05g, 69.6mmol) in dichloromethane (50mL), add methanesulfonate at 0°C Chlorine (7.98 g, 69.6 mmol). The reaction solution was slowly raised to room temperature and stirred for 2 hours. The reaction was quenched by the addition of aqueous sodium bicarbonate (50 mL). Extract with dichloromethane (20 mL x 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain methyl trans-4-methanesulfonyloxymethyl-cyclohexanecarboxylate (5.80g, yellow Oily), yield: 99%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 4.10-4.03 (m, 2H), 3.65 (s, 3H), 3.07 (s, 3H), 2.42-2.31 (m, 1H), 2.10-2.03 ( m, 2H), 1.90-1.82 (m, 2H), 1.75-1.66 (m, 1H), 1.48-1.42 (m, 2H), 1.21-1.10 (m, 2H). MS-ESI calculated value [M+H] + 251, measured value 251.

第三步。 third step.

反式-甲基4-[(3，7-二甲基-2，6-二氧代-嘌呤-1-基)甲基]環己烷羧酸。 Trans-Methyl 4-[(3,7-dimethyl-2,6-dioxo-purin-1-yl)methyl]cyclohexanecarboxylic acid.

將反式-4-甲磺醯氧基甲基-環己烷羧酸甲酯(1.00g，4.00mmol)，3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(719mg，4.00mmol)，碘化鉀(66.0mg，0.397mmol)和碳酸鉀(1.10g，7.96mmol)溶於N，N-二甲基甲醯胺(10mL)中。反應液升溫至120℃，攪拌3小時。冷却至室溫，過濾，濾液減壓濃縮。用矽膠柱色譜法分離純化(乙酸乙酯，Rf值=0.1)，得到反式-甲基4-[(3，7-二甲基-2，6-二氧代-嘌呤-1-基)甲基]環己烷羧酸(800mg，黃色固體)，產率：60%。1H NMR：(400MHz，Methonal-d 4)δ7.88(s，1H)，3.98(s，3H)，3.90-3.82(m，2H)，3.72(s，3H)，3.51(s，3H)，2.33-2.25(m，1H)，2.03-1.98(m，2H)，1.80-1.74(m，3H)，1.42-1.36(m，2H)，1.21-1.10(m，2H)。MS-ESI計算值[M+H]+ 335，實測值335。 The trans-4-methylsulfonyloxymethyl-cyclohexanecarboxylic acid methyl ester (1.00g, 4.00mmol), 3,7-dimethyl- 1H -purine-2,6( 3H ,7 H )-diketone (719 mg, 4.00 mmol), potassium iodide (66.0 mg, 0.397 mmol) and potassium carbonate (1.10 g, 7.96 mmol) were dissolved in N , N -dimethylformamide (10 mL). The reaction solution was heated to 120°C and stirred for 3 hours. Cool to room temperature, filter, and concentrate the filtrate under reduced pressure. Separated and purified by silica gel column chromatography (ethyl acetate, Rf value=0.1) to obtain trans-methyl 4-[(3,7-dimethyl-2,6-dioxo-purin-1-yl) Methyl]cyclohexanecarboxylic acid (800 mg, yellow solid), yield: 60%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.88 (s, 1H), 3.98 (s, 3H), 3.90-3.82 (m, 2H), 3.72 (s, 3H), 3.51 (s, 3H) , 2.33-2.25 (m, 1H), 2.03-1.98 (m, 2H), 1.80-1.74 (m, 3H), 1.42-1.36 (m, 2H), 1.21-1.10 (m, 2H). MS-ESI calculated value [M+H] + 335, measured value 335.

第四步。 the fourth step.

1-(反式-4-乙醯基環己基甲基)-3，7-二甲基-3，7-二氫-嘌呤-2，6-二酮 1-(trans-4-acetylcyclohexylmethyl)-3,7-dimethyl-3,7-dihydro-purine-2,6-dione

將反式-甲基4-[(3，7-二甲基-2，6-二氧代-嘌呤-1-基)甲基]環己烷羧酸(300mg，0.897mmol)和O，N-二甲基羥胺鹽酸鹽(114mg，1.17mmol)溶於四氫呋喃(25mL)中，0℃下加入甲基溴化鎂(3M乙醚溶液，1.50mL，4.50mmol)。反應液緩慢升至室溫，攪拌12小時。加入飽和氯化銨(10mL)淬滅反應。用乙酸乙酯萃取(10mL x 3)。合併有機相，用飽和食鹽水(30mL)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用製備TLC板純化(乙酸乙酯，Rf=0.4)，得到1-(反式-4-乙醯基環己基甲基)-3，7-二甲基-3，7-二氫-嘌呤-2，6-二酮(80.0mg，黃色油狀)，產率：29%。1H NMR：(400MHz，Methonal-d 4 )δ7.88(s，1H)，3.98(s，3H)，3.92-3.84(m，2H)，3.55(s，3H)，2.42-2.33(m，1H)，2.15(s，3H)，1.98-1.88(m，2H)，1.85-1.75(m，3H)，1.32-1.10(m，4H)。MS-ESI計算值[M+H]+ 319，實測值319。 Combine trans-methyl 4-[(3,7-dimethyl-2,6-dioxo-purin-1-yl)methyl]cyclohexanecarboxylic acid (300mg, 0.897mmol) and O , N -Dimethylhydroxylamine hydrochloride (114mg, 1.17mmol) was dissolved in tetrahydrofuran (25mL), and methylmagnesium bromide (3M ether solution, 1.50mL, 4.50mmol) was added at 0°C. The reaction solution was slowly raised to room temperature and stirred for 12 hours. The reaction was quenched by adding saturated ammonium chloride (10 mL). Extract with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purified with a preparative TLC plate (ethyl acetate, Rf=0.4) to obtain 1-(trans-4-acetylcyclohexylmethyl)-3,7-dimethyl-3,7-dihydro-purine- 2,6-Diketone (80.0 mg, yellow oil), yield: 29%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.88 (s, 1H), 3.98 (s, 3H), 3.92-3.84 (m, 2H), 3.55 (s, 3H), 2.42-2.33 (m, 1H), 2.15 (s, 3H), 1.98-1.88 (m, 2H), 1.85-1.75 (m, 3H), 1.32-1.10 (m, 4H). MS-ESI calculated value [M+H] + 319, measured value 319.

第五步。 the fifth step.

3，7-二甲基-1-[[反式-4-(2，2，2-三氟-1-羥基-1-甲基-乙基)環己基]甲基]嘌呤-2，6-二酮。 3,7-Dimethyl-1-[[trans-4-(2,2,2-trifluoro-1-hydroxy-1-methyl-ethyl)cyclohexyl]methyl]purine-2,6 -Diketone.

將1-(反式-4-乙醯基環己基甲基)-3，7-二甲基-3，7-二氫-嘌呤-2，6-二酮(80.0mg，0.251mmol)，氟化銫(11.5mg，0.753mmol)溶於四氫呋喃(10mL)中，室溫下加入三甲基-三氟甲基-矽烷(71.6mg，0.502mmol)，攪拌12小時。加入1N鹽酸(10mL)室溫下攪拌1小時，加入飽和碳酸氫鈉(50mL)淬滅反應。用乙酸乙酯萃取(10mL x 3)。合併有機相，用飽和食鹽水(30mL)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃 縮。加入用製備型高效液相色譜純化，得到3，7-二甲基-1-[[反式-4-(2，2，2-三氟-1-羥基-1-甲基-乙基)環己基]甲基]嘌呤-2，6-二酮(35.0mg，黃色固體)，產率：70%。1H NMR：(400MHz，Methonal-d 4)δ7.88(s，1H)，3.98(s，3H)，3.88(d，J=6.8Hz，2H)，3.53(s，3H)，1.96-1.67(m，6H)，1.22(s，3H)，1.15-1.06(m，4H)。MS-ESI計算值[M+H]+ 389，實測值389。 Add 1-(trans-4-acetylcyclohexylmethyl)-3,7-dimethyl-3,7-dihydro-purine-2,6-dione (80.0mg, 0.251mmol), fluorine Cesium chloride (11.5 mg, 0.753 mmol) was dissolved in tetrahydrofuran (10 mL), trimethyl-trifluoromethyl-silane (71.6 mg, 0.502 mmol) was added at room temperature, and the mixture was stirred for 12 hours. 1N hydrochloric acid (10 mL) was added and stirred at room temperature for 1 hour, and saturated sodium bicarbonate (50 mL) was added to quench the reaction. Extract with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purify by preparative high performance liquid chromatography to obtain 3,7-dimethyl-1-[[trans-4-(2,2,2-trifluoro-1-hydroxy-1-methyl-ethyl) Cyclohexyl]methyl]purine-2,6-dione (35.0 mg, yellow solid), yield: 70%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.88 (s, 1H), 3.98 (s, 3H), 3.88 (d, J = 6.8 Hz, 2H), 3.53 (s, 3H), 1.96-1.67 (m, 6H), 1.22 (s, 3H), 1.15-1.06 (m, 4H). MS-ESI calculated value [M+H] + 389, measured value 389.

實施例20。 Example 20.

3，7-二甲基-1-[反式-4-(2，2，2-三氟-1-羥基-1-三氟甲基-乙基)-環己基甲基]-3，7-二氫-嘌呤-2，6-二酮。 3,7-Dimethyl-1-[trans-4-(2,2,2-Trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-cyclohexylmethyl]-3,7 -Dihydro-purine-2,6-dione.

Figure 105135042-A0305-02-0080-81
Figure 105135042-A0305-02-0080-81

第一步。 first step.

3，7-二甲基-1-[反式-4-(2，2，2-三氟-1-羥基-1-三氟甲基-乙基)-環己基甲基]-3，7-二氫-嘌呤-2，6-二酮。 3,7-Dimethyl-1-[trans-4-(2,2,2-Trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-cyclohexylmethyl]-3,7 -Dihydro-purine-2,6-dione.

將反式-甲基4-[(3，7-二甲基-2，6-二氧代-嘌呤-1-基)甲基]環己烷羧酸(200mg，0.598mmol)，氟化銫(45.4mg，0.299mmol)溶於四氫呋喃(10mL)中，室溫下加入三甲基-三氟甲基-矽烷(340mg，2.39mmol)，攪拌12小時。加入1N鹽酸(10mL)室溫下攪拌1小時，加入飽和碳酸氫鈉(50mL)淬滅反應。用乙酸乙酯萃取(10mL x 3)。合併有機相，用飽和食鹽水(20mL)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。加入用製備型高效液相色譜純化，得到3，7-二甲基-1-[反式-4-(2，2， 2-三氟-1-羥基-1-三氟甲基-乙基)-環己基甲基]-3，7-二氫-嘌呤-2，6-二酮(35.0mg，黃色固體)，產率：41%。 Trans-methyl 4-[(3,7-dimethyl-2,6-dioxo-purin-1-yl)methyl]cyclohexanecarboxylic acid (200mg, 0.598mmol), cesium fluoride (45.4 mg, 0.299 mmol) was dissolved in tetrahydrofuran (10 mL), trimethyl-trifluoromethyl-silane (340 mg, 2.39 mmol) was added at room temperature, and the mixture was stirred for 12 hours. 1N hydrochloric acid (10 mL) was added and stirred at room temperature for 1 hour, and saturated sodium bicarbonate (50 mL) was added to quench the reaction. Extract with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. It was purified by preparative high performance liquid chromatography to obtain 3,7-dimethyl-1-[trans-4-(2,2, 2-Trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-cyclohexylmethyl]-3,7-dihydro-purine-2,6-dione (35.0mg, yellow solid), produced Rate: 41%.

1H NMR：(400MHz，Methonal-d 4)δ7.88(s，1H)，3.98(s，3H)，3.88(d，J=6.8Hz，2H)，3.53(s，3H)，2.08-1.79(m，6H)，1.30-1.24(m，2H)，1.11-1.08(m，2H)。MS-ESI計算值[M+H]+ 443，實測值443。 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.88 (s, 1H), 3.98 (s, 3H), 3.88 (d, J = 6.8 Hz, 2H), 3.53 (s, 3H), 2.08-1.79 (m, 6H), 1.30-1.24 (m, 2H), 1.11-1.08 (m, 2H). MS-ESI calculated value [M+H] + 443, measured value 443.

實施例21。 Example 21.

1-[[反式-4-(1-羥基環丙基)環己基]甲基]-3，7-二甲基嘌呤-2，6-二酮。 1-[[trans-4-(1-hydroxycyclopropyl)cyclohexyl]methyl]-3,7-dimethylpurine-2,6-dione.

Figure 105135042-A0305-02-0081-83
Figure 105135042-A0305-02-0081-83

第一步。 first step.

1-[[反式-4-(1-羥基環丙基)環己基]甲基]-3，7-二甲基嘌呤-2，6-二酮。 1-[[trans-4-(1-hydroxycyclopropyl)cyclohexyl]methyl]-3,7-dimethylpurine-2,6-dione.

將反式-甲基4-[(3，7-二甲基-2，6-二氧代-嘌呤-1-基)甲基]環己烷羧酸(200mg，0.598mmol)，四異丙基氧化鈦(340mg，1.20mmol)溶於四氫呋喃(10mL)中，室溫下加入乙基溴化鎂(3M乙醚溶液，0.39mL，1.17mmol)，攪拌12小時。加入飽和氯化銨(50mL)淬滅反應。用乙酸乙酯萃取(10mL x 3)。合併有機相，用飽和食鹽水(30mL)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。剩餘物用製備型高效液相色譜純化，得到1-[[反式-4-(1-羥基環丙基)環己基]甲基]-3，7-二甲基嘌呤-2，6-二酮(70.0mg，黃色固體)，產率：35%。 Trans-methyl 4-[(3,7-dimethyl-2,6-dioxo-purin-1-yl)methyl]cyclohexanecarboxylic acid (200mg, 0.598mmol), tetraisopropyl Titanium oxide (340 mg, 1.20 mmol) was dissolved in tetrahydrofuran (10 mL), ethyl magnesium bromide (3M ether solution, 0.39 mL, 1.17 mmol) was added at room temperature, and the mixture was stirred for 12 hours. The reaction was quenched by adding saturated ammonium chloride (50 mL). Extract with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative high performance liquid chromatography to obtain 1-[[trans-4-(1-hydroxycyclopropyl)cyclohexyl]methyl]-3,7-dimethylpurine-2,6-di Ketone (70.0 mg, yellow solid), yield: 35%.

1H NMR：(400MHz，Methonal-d 4 )δ7.87(s，1H)，3.98(s，3H)，3.88(d，J=6.8Hz，2H)，3.53(s，3H)，1.79-1.71(m，5H)，1.29-1.07(m，5H)，0.60-0.57(m，2H)，0.42-0.39(m，2H)。MS-ESI計算值[M+H]+ 333，實測值333。 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.87 (s, 1H), 3.98 (s, 3H), 3.88 (d, J = 6.8 Hz, 2H), 3.53 (s, 3H), 1.79-1.71 (m, 5H), 1.29-1.07 (m, 5H), 0.60-0.57 (m, 2H), 0.42-0.39 (m, 2H). MS-ESI calculated value [M+H] + 333, measured value 333.

實施例22。 Example 22.

1-(2-(3-乙基-3-羥基環己基)乙基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1- (2- (3-ethyl-3-hydroxy cyclohexyl) ethyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

Figure 105135042-A0305-02-0082-84
Figure 105135042-A0305-02-0082-84

第一步。 first step.

2-(3-乙基-3-羥基環己基)乙基甲磺酸酯。 2-(3-Ethyl-3-hydroxycyclohexyl)ethyl methanesulfonate.

將1-乙基-3-(2-羥基乙基)環己醇(450mg，2.61mmol)及二異丙基乙基胺(500mg，3.92mmol)溶於二氯甲烷(10mL)中，在0℃下緩慢加入甲烷磺醯氯(600mg，5.40mmol)。反應液在0℃攪拌0.5小時。加入水淬滅反應，用乙酸乙酯萃取(20mL x 3)。合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮用矽膠柱色譜法純化(10：1石油醚/乙酸乙酯，Rf=0.4)得到產物2-(3-乙基-3-羥基環己基)乙基甲磺酸酯(450mg，黃色油狀物)，產率：69%。MS-ESI計算值[M+H]+ 251，實測值251。 1-Ethyl-3-(2-hydroxyethyl)cyclohexanol (450mg, 2.61mmol) and diisopropylethylamine (500mg, 3.92mmol) were dissolved in dichloromethane (10mL) and heated at 0 Slowly add methanesulfonyl chloride (600mg, 5.40mmol) at °C. The reaction solution was stirred at 0°C for 0.5 hour. The reaction was quenched by adding water and extracted with ethyl acetate (20 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (10:1 petroleum ether/ethyl acetate, Rf=0.4) to obtain the product 2-(3-ethyl-3-hydroxyl Cyclohexyl) ethyl methanesulfonate (450 mg, yellow oil), yield: 69%. MS-ESI calculated value [M+H] + 251, measured value 251.

第二步。 The second step.

1-(2-(3-乙基-3-羥基環己基)乙基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1- (2- (3-ethyl-3-hydroxy cyclohexyl) ethyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

將2-(3-乙基-3-羥基環己基)乙基甲磺酸酯(200mg，0.790mmol)，3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(144mg，0.790mmol)及碳酸鉀(220mg，1.60mmol)，碘化鉀(13.1mg，0.0790mmol)溶於N，N-二甲基甲醯胺(3mL)中。反應液加熱至130℃，攪拌3小時。反應液冷却至25℃，加入飽和食鹽水，用乙酸乙酯萃取(40mL x 3)。合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用製備高效液相色譜分離純化得到產物1-(2-(3-乙基-3-羥基環己基)乙基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(70.0mg，白色固體)，產率：26%。1H NMR：(400MHz，Methonal-d 4 )δ7.86(s，1H)，4.09-3.94(m，5H)，3.52(s，3H)，1.88-1.84(m，1H)，1.80-1.40(m，10H)，1.26-1.16(m，1H)，1.02-0.95(m，1H)，0.91(t，J=7.2Hz，3H)。MS-ESI計算值[M+H-18]+ 317，實測值317。 2- (3-ethyl-3-hydroxy-cyclohexyl) ethyl methanesulfonate (200mg, 0.790mmol), 3,7- dimethyl -1 H - purine -2,6 (3 H, 7 H )-Diketone (144mg, 0.790mmol) and potassium carbonate (220mg, 1.60mmol), potassium iodide (13.1mg, 0.0790mmol) was dissolved in N , N -dimethylformamide (3mL). The reaction solution was heated to 130°C and stirred for 3 hours. The reaction solution was cooled to 25°C, saturated brine was added, and extracted with ethyl acetate (40 mL x 3). The organic phases were combined, dried with anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and separated and purified by preparative high performance liquid chromatography to obtain the product 1-(2-(3-ethyl-3-hydroxycyclohexyl)ethyl)-3, 7-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (70.0 mg, white solid), yield: 26%. 1 H NMR : (400MHz, Methonal- d 4 ) δ 7.86 (s, 1H), 4.09-3.94 (m, 5H), 3.52 (s, 3H), 1.88-1.84 (m, 1H), 1.80-1.40 ( m, 10H), 1.26-1.16 (m, 1H), 1.02-0.95 (m, 1H), 0.91 (t, J = 7.2 Hz, 3H). MS-ESI calculated value [M+H-18] + 317, measured value 317.

實施例23。 Example 23.

Figure 105135042-A0305-02-0083-86
Figure 105135042-A0305-02-0083-86
Figure 105135042-A0305-02-0084-87
Figure 105135042-A0305-02-0084-87

第一步。 first step.

3-三氟甲基-3-三甲基矽烷氧基-環己烷甲酸乙酯。 3-Trifluoromethyl-3-trimethylsilyloxy-cyclohexane ethyl ester.

將乙基-3-氧代環己烷羧酸(1.00g，5.88mmol)，氟化銫(446mg，2.94mmol)溶於四氫呋喃(30mL)中，室溫下加入三甲基-三氟甲基-矽烷(1.67g，11.7mmol)，攪拌12小時。加入水(20mL)淬滅反應。用乙酸乙酯萃取(20mL x 3)。合併有機相，用飽和食鹽水(60mL)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到3-三氟甲基-3-三甲基矽烷氧基-環己烷甲酸乙酯(1.40g，黃色油狀)，產率：76%。MS-ESI計算值[M+H]+ 313，實測值313。 Dissolve ethyl-3-oxocyclohexanecarboxylic acid (1.00g, 5.88mmol) and cesium fluoride (446mg, 2.94mmol) in tetrahydrofuran (30mL), add trimethyl-trifluoromethyl at room temperature -Silane (1.67g, 11.7mmol), stir for 12 hours. The reaction was quenched by adding water (20 mL). Extract with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine (60 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain ethyl 3-trifluoromethyl-3-trimethylsilyloxy-cyclohexanecarboxylate (1.40g , Yellow oil), yield: 76%. MS-ESI calculated value [M+H] + 313, measured value 313.

第二步。 The second step.

(3-三氟甲基-3-三甲基矽烷氧基環己基)甲醇。 (3-Trifluoromethyl-3-trimethylsilyloxycyclohexyl)methanol.

將3-三氟甲基-3-三甲基矽烷氧基-環己烷甲酸乙酯(1.00g，3.20mmol)溶於四氫呋喃(10mL)中，0℃下，加入四氫鋰鋁(243mg，6.40mmol)，反應1小時。加入水(10mL)淬滅反應。用乙酸乙酯萃取(20mL x 3)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到(3-三氟甲基-3-三甲基 矽烷氧基環己基)甲醇(800mg，無色油狀)，產率：92%。MS-ESI計算值[M+H]+ 271，實測值271。 3-Trifluoromethyl-3-trimethylsilyloxy-cyclohexane ethyl ester (1.00g, 3.20mmol) was dissolved in tetrahydrofuran (10mL), at 0°C, tetrahydrolithium aluminum (243mg, 6.40mmol), react for 1 hour. The reaction was quenched by adding water (10 mL). Extract with ethyl acetate (20mL x 3), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain (3-trifluoromethyl-3-trimethylsilyloxycyclohexyl)methanol (800mg, colorless oil) , Yield: 92%. MS-ESI calculated value [M+H] + 271, measured value 271.

第三步。 third step.

[3-(三氟甲基)-3-三甲基矽氧基環己基]甲基甲磺酸酯。 [3-(Trifluoromethyl)-3-trimethylsiloxycyclohexyl] methanesulfonate.

將3-三氟甲基-3-三甲基矽烷氧基環己基)甲醇(850mg，3.14mmol)和三乙胺(953mg，9.42mmol)溶於二氯甲烷(15mL)中，0℃下加入甲烷磺醯氯(719mg，6.28mmol)。反應液緩慢升至室溫，攪拌2小時。加入碳酸氫鈉水溶液(10mL)淬滅反應。用二氯甲烷萃取(20mL x 3)。合併有機相，用飽和食鹽水(20mL)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到[3-(三氟甲基)-3-三甲基矽氧基環己基]甲基甲磺酸酯(900mg，黃色油狀)，產率：82%。1H NMR：(400MHz，Methonal-d 4 )δ4.36-4.32(m，1H)，4.17-4.13(m，1H)，3.08(s，3H)，2.12-1.60(m，9H)，0.16(s，9H)。MS-ESI計算值[M+H]+ 349，實測值349。 Dissolve 3-trifluoromethyl-3-trimethylsilyloxycyclohexyl)methanol (850mg, 3.14mmol) and triethylamine (953mg, 9.42mmol) in dichloromethane (15mL) and add at 0°C Methanesulfonyl chloride (719 mg, 6.28 mmol). The reaction solution was slowly raised to room temperature and stirred for 2 hours. The reaction was quenched by the addition of aqueous sodium bicarbonate (10 mL). Extract with dichloromethane (20 mL x 3). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain [3-(trifluoromethyl)-3-trimethylsiloxycyclohexyl]methanesulfonate Ester (900mg, yellow oil), yield: 82%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 4.36-4.32 (m, 1H), 4.17-4.13 (m, 1H), 3.08 (s, 3H), 2.12-1.60 (m, 9H), 0.16 ( s, 9H). MS-ESI calculated value [M+H] + 349, measured value 349.

第四步。 the fourth step.

3，7-二甲基-1-[[3-(三氟甲基)-3-三甲基矽氧基-環己基]甲基]嘌呤-2，6-二酮。 3,7-Dimethyl-1-[[3-(trifluoromethyl)-3-trimethylsiloxy-cyclohexyl]methyl]purine-2,6-dione.

將[3-(三氟甲基)-3-三甲基矽氧基環己基]甲基甲磺酸酯(200mg，0.573mmol)，3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(103mg，0.574mmol)，碘化鉀(28.6mg，0.172mmol)和碳酸鉀(374mg，1.15mmol)溶於N，N-二甲基甲醯胺(30mL)中。反應液升溫至120℃，攪拌3小時。冷却至室溫，過濾，濾液減壓濃縮用製備型高效液相色譜純化，得到3，7-二甲基-1-[[3-(三氟甲基)-3-三甲基矽氧基-環己基]甲基]嘌呤-2，6-二酮(150mg，黃色固體)，產率：60%。 [3-(Trifluoromethyl)-3-trimethylsiloxycyclohexyl]methanesulfonate (200mg, 0.573mmol), 3,7-dimethyl- 1H -purine-2, 6 (3 H, 7 H) - dione (103mg, 0.574mmol), potassium iodide (28.6mg, 0.172mmol) and potassium carbonate (374mg, 1.15mmol) was dissolved in N, N - dimethylformamide (30mL) in. The reaction solution was heated to 120°C and stirred for 3 hours. Cooled to room temperature, filtered, the filtrate was concentrated under reduced pressure and purified by preparative high performance liquid chromatography to obtain 3,7-dimethyl-1-[[3-(trifluoromethyl)-3-trimethylsiloxy -Cyclohexyl]methyl]purine-2,6-dione (150 mg, yellow solid), yield: 60%.

MS-ESI計算值[M+H]+ 433，實測值433。 MS-ESI calculated value [M+H] + 433, measured value 433.

第五步。 the fifth step.

將3，7-二甲基-1-[[3-(三氟甲基)-3-三甲基矽氧基-環己基]甲基]嘌呤-2，6-二酮(200mg，0.462mmol)溶於四氫呋喃(10mL)中，加入1N鹽酸(10mL)室溫下攪拌1小時，加入飽和碳酸氫鈉(50mL)淬滅反應。用乙酸乙酯萃取(20mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用製備型高效液相色譜純化，得到產物1(10.0mg，黃色固體)(異構體1，第一個峰)，產率：6%。1H NMR：(400MHz，Methonal-d 4 )δ7.87(s，1H)，3.97(s，3H)，3.89-3.83(m，2H)，3.52(s，3H)，2.23-2.22(m，1H)，1.76-1.07(m，8H)。MS-ESI計算值[M+H]+ 361，實測值361。 Add 3,7-dimethyl-1-[[3-(trifluoromethyl)-3-trimethylsiloxy-cyclohexyl]methyl]purine-2,6-dione (200mg, 0.462mmol ) Was dissolved in tetrahydrofuran (10 mL), 1N hydrochloric acid (10 mL) was added and stirred at room temperature for 1 hour, and saturated sodium bicarbonate (50 mL) was added to quench the reaction. Extract with ethyl acetate (20 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purified by preparative high performance liquid chromatography to obtain product 1 (10.0 mg, yellow solid) (isomer 1, first peak), yield: 6%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.87 (s, 1H), 3.97 (s, 3H), 3.89-3.83 (m, 2H), 3.52 (s, 3H), 2.23-2.22 (m, 1H), 1.76-1.07 (m, 8H). MS-ESI calculated value [M+H] + 361, measured value 361.

產物2(85.0mg黃色固體)(異構體2，第二個峰)，產率：51%。1H NMR：(400MHz，Methonal-d 4 )δ7.87(s，1H)，4.31-4.26(m，1H)，3.99-3.95(m，4H)，3.55(s，3H)，2.26-1.88(m，3H)，1.79-1.47(m，6H)。MS-ESI計算值[M+H]+ 361，實測值361。 Product 2 (85.0 mg yellow solid) (isomer 2, second peak), yield: 51%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.87 (s, 1H), 4.31-4.26 (m, 1H), 3.99-3.95 (m, 4H), 3.55 (s, 3H), 2.26-1.88 ( m, 3H), 1.79-1.47 (m, 6H). MS-ESI calculated value [M+H] + 361, measured value 361.

實施例24。 Example 24.

1-((5-羥基-5-(三氟甲基)四氫-2H-吡喃-2-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((5-hydroxy-5-(trifluoromethyl)tetrahydro- 2H -pyran-2-yl)methyl)-3,7-dimethyl- 1H -purine-2,6- (3 H , 7 H )-dione.

Figure 105135042-A0305-02-0086-88
Figure 105135042-A0305-02-0086-88
Figure 105135042-A0305-02-0087-89
Figure 105135042-A0305-02-0087-89

第一步。 first step.

(3，4-二氫-2H-吡喃-2-基)甲醇。 (3,4-Dihydro- 2H -pyran-2-yl)methanol.

將3，4-二氫-2H-吡喃-2-甲醛(3.00g，26.7mmol)溶於甲醇(20mL)中，0℃下加入硼氫化鈉(2.02g，53.5mmol)，反應2小時。加入飽和氯化銨(30mL)淬滅反應。用二氯甲烷萃取(20mL x 3)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到(3，4-二氫-2H-吡喃-2-基)甲醇(1.50g，黃色油狀)，產率：49%。1H NMR：(400MHz，Methonal-d 4 )δ6.40(d，J=6.0Hz，1H)，4.71-4.68(m，1H)，3.86-3.83(m，1H)，3.82-3.61(m，2H)，2.13-2.12(m，1H)，2.10-2.08(m，1H)，2.02-2.01(m，1H)，1.68-1.63(m，1H)。 Dissolve 3,4-dihydro- 2H -pyran-2-carbaldehyde (3.00g, 26.7mmol) in methanol (20mL), add sodium borohydride (2.02g, 53.5mmol) at 0°C, and react for 2 hours . The reaction was quenched by adding saturated ammonium chloride (30 mL). Extract with dichloromethane (20mL x 3), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain (3,4-dihydro- 2H -pyran-2-yl)methanol (1.50g, yellow oil) , Yield: 49%. 1 H NMR: (400MHz, Methonal- d 4 ) δ6.40 (d, J = 6.0Hz, 1H), 4.71-4.68 (m, 1H), 3.86-3.83 (m, 1H), 3.82-3.61 (m, 2H), 2.13-2.12 (m, 1H), 2.10-2.08 (m, 1H), 2.02-2.01 (m, 1H), 1.68-1.63 (m, 1H).

第二步。 The second step.

(3，4-二氫-2H-吡喃-2-基)甲基甲磺酸酯。 (3,4-Dihydro- 2H -pyran-2-yl)methanesulfonate.

將(3，4-二氫-2H-吡喃-2-基)甲醇(1.50g，13.1mmol)和三乙胺(2.66g，26.3mmol)溶於二氯甲烷(20mL)中，0℃下加入甲烷磺醯氯(3.01g，26.3mmol)。反應液緩慢升至室溫，攪拌2小時。加入碳酸氫鈉水溶液(10mL)淬滅反應。用二氯甲烷萃取(20mL x 3)。合併有機 相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到(3，4-二氫-2H-吡喃-2-基)甲基甲磺酸酯(1.70g，黃色油狀)，產率：67%。MS-ESI計算值[M+H]+ 193，實測值193。 (3,4-Dihydro- 2H -pyran-2-yl)methanol (1.50g, 13.1mmol) and triethylamine (2.66g, 26.3mmol) were dissolved in dichloromethane (20mL) at 0°C Add methanesulfonyl chloride (3.01g, 26.3mmol). The reaction solution was slowly raised to room temperature and stirred for 2 hours. The reaction was quenched by the addition of aqueous sodium bicarbonate (10 mL). Extract with dichloromethane (20 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain (3,4-dihydro- 2H -pyran-2-yl)methanesulfonate (1.70g, Yellow oil), yield: 67%. MS-ESI calculated value [M+H] + 193, measured value 193.

第三步。 third step.

1-((3，4-二氫-2H-吡喃-2-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((3,4-Dihydro-2 H -pyran-2-yl)methyl)-3,7-dimethyl-1 H -purine-2,6-(3 H ,7 H )- Diketone.

將(3，4-二氫-2H-吡喃-2-基)甲基甲磺酸酯(1.70g，8.84mmol)，3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(1.59g，8.84mmol)，碘化鉀(146mg，0.884mmol)和碳酸鉀(2.44g，17.7mmol)溶於N，N-二甲基甲醯胺(50mL)中。反應液升溫至120℃，攪拌3小時。冷却至室溫，過濾，濾液減壓濃縮用矽膠柱色譜法純化(乙酸乙酯，Rf=0.4)，得到1-((3，4-二氫-2H-吡喃-2-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(1.30g，黃色固體)，產率：53%。MS-ESI計算值[M+H]+ 277，實測值277。 Add (3,4-dihydro- 2H -pyran-2-yl)methanesulfonate (1.70g, 8.84mmol), 3,7-dimethyl- 1H -purine-2,6- (3 H , 7 H )-dione (1.59g, 8.84mmol), potassium iodide (146mg, 0.884mmol) and potassium carbonate (2.44g, 17.7mmol) dissolved in N , N -dimethylformamide (50mL) in. The reaction solution was heated to 120°C and stirred for 3 hours. Cooled to room temperature, filtered, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (ethyl acetate, Rf=0.4) to obtain 1-((3,4-dihydro-2 H -pyran-2-yl)methane yl) -3,7-dimethyl -1 H - purine -2,6- (3 H, 7 H) - dione (1.30 g, yellow solid), yield: 53%. MS-ESI calculated value [M+H] + 277, measured value 277.

第四步。 the fourth step.

1-((5-羥基四氫-2H-吡喃-2-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((5-hydroxytetrahydro- 2H -pyran-2-yl)methyl)-3,7-dimethyl- 1H -purine-2,6-( 3H , 7H )-di ketone.

將1-(3，4-二氫-2H-吡喃-2-基甲基)-3，7-二甲基嘌呤-2，6-二酮(600mg，2.17mmol)溶於四氫呋喃(30mL)中，0℃下加入硼烷二甲硫醚(825mg，10.7mmol)。反應液緩慢升至室溫，攪拌12小時。加入3N氫氧化鈉水溶液(30mL)和雙氧水(10mL)，繼續反應1小時。加入甲醇(10mL)淬滅反應，硫代硫酸鈉溶液(30mL)洗滌，用二氯甲烷萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用製備TLC板分離純化(20：1二氯甲烷/甲醇，Rf=0.3) 得到1-((5-羥基四氫-2H-吡喃-2-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(130mg，黃色油狀)，產率：20%。1H NMR：(400MHz，Methonal-d 4 )δ7.88(s，1H)，4.25-4.23(m，1H)，4.20(s，3H)，3.98-3.67(m，5H)，3.54(s，3H)，2.10-1.77(m，2H)，1.49-1.31(m，2H)。MS-ESI計算值[M+H]+ 295，實測值295。 Dissolve 1-(3,4-dihydro- 2H -pyran-2-ylmethyl)-3,7-dimethylpurine-2,6-dione (600mg, 2.17mmol) in tetrahydrofuran (30mL ), borane dimethyl sulfide (825 mg, 10.7 mmol) was added at 0°C. The reaction solution was slowly raised to room temperature and stirred for 12 hours. 3N aqueous sodium hydroxide solution (30 mL) and hydrogen peroxide (10 mL) were added, and the reaction was continued for 1 hour. The reaction was quenched by adding methanol (10 mL), washed with sodium thiosulfate solution (30 mL), and extracted with dichloromethane (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Separate and purify with a preparative TLC plate (20:1 dichloromethane/methanol, Rf=0.3) to obtain 1-((5-hydroxytetrahydro- 2H -pyran-2-yl)methyl)-3,7-di Methyl- 1H -purine-2,6-( 3H , 7H )-dione (130mg, yellow oil), yield: 20%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.88 (s, 1H), 4.25-4.23 (m, 1H), 4.20 (s, 3H), 3.98-3.67 (m, 5H), 3.54 (s, 3H), 2.10-1.77 (m, 2H), 1.49-1.31 (m, 2H). MS-ESI calculated value [M+H] + 295, measured value 295.

第五步。 the fifth step.

3，7-二甲基-1-((5-氧代四氫-2H-吡喃-2-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3,7-dimethyl-1 - ((5-oxo-tetrahydro -2 H - pyran-2-yl) methyl) -1 H - purine -2,6- (3 H, 7 H) - Diketone.

將1-((5-羥基四氫-2H-吡喃-2-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(130mg，0.441mmol)溶於二氯甲烷(10mL)中，加入戴斯馬丁過碘烷(138mg，1.33mmol)，25℃反應3小時。加入飽和硫代硫酸鈉溶液(20mL)淬滅反應，二氯甲烷(10mL x 3)萃取，合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用製備TLC板分離純化(20：1二氯甲烷/甲醇，Rf=0.4)，得到3，7-二甲基-1-((5-氧代四氫-2H-吡喃-2-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮(60.0mg，黃色固體)，產率：47%。MS-ESI計算值[M+H]+ 293，實測值293。 The 1-((5-hydroxytetrahydro- 2H -pyran-2-yl)methyl)-3,7-dimethyl- 1H -purine-2,6-( 3H , 7H )- The diketone (130 mg, 0.441 mmol) was dissolved in dichloromethane (10 mL), and Dessmartin periodinane (138 mg, 1.33 mmol) was added, and the reaction was carried out at 25°C for 3 hours. The reaction was quenched by adding saturated sodium thiosulfate solution (20 mL), extracted with dichloromethane (10 mL x 3), and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Separate and purify with a preparative TLC plate (20:1 dichloromethane/methanol, Rf=0.4) to obtain 3,7-dimethyl-1-((5-oxotetrahydro-2H-pyran-2-yl) methyl) -1 H - purine -2,6- (3 H, 7 H) - dione (60.0 mg, yellow solid), yield: 47%. MS-ESI calculated value [M+H] + 293, measured value 293.

第六步。 The sixth step.

1-((5-羥基-5-(三氟甲基)四氫-2H-吡喃-2-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((5-hydroxy-5-(trifluoromethyl)tetrahydro- 2H -pyran-2-yl)methyl)-3,7-dimethyl- 1H -purine-2,6- (3 H , 7 H )-dione.

將3，7-二甲基-1-((5-氧代四氫-2H-吡喃-2-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮(60.0mg，0.205mmol)，氟化銫(6.24mg，0.0411mmol)溶於四氫呋喃(10mL)中，室溫下加入三甲基三氟甲基矽烷(87.5mg，0.615mmol)，攪拌5小時。加入1N鹽酸(10mL)室溫下攪拌1小時， 加入飽和碳酸氫鈉(50mL)淬滅反應。用乙酸乙酯萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用製備型高效液相色譜純化，得到1-((5-羥基-5-(三氟甲基)四氫-2H-吡喃-2-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(15.0mg，黃色固體)，產率：30%。1H NMR：(400MHz，Methonal-d 4 )δ8.28(s，1H)，4.39-4.10(m，2H)，4.05(s，3H)，3.93-3.89(m，2H)，3.55(s，3H)，3.32-3.27(m，1H)，1.89-1.65(m，4H)。MS-ESI計算值[M+H]+ 363，實測值363。 3,7-Dimethyl-1-((5-oxotetrahydro- 2H -pyran-2-yl)methyl)-1H-purine-2,6-( 3H , 7H )- Dione (60.0mg, 0.205mmol), cesium fluoride (6.24mg, 0.0411mmol) were dissolved in tetrahydrofuran (10mL), added trimethyltrifluoromethylsilane (87.5mg, 0.615mmol) at room temperature, and stirred for 5 hour. 1N hydrochloric acid (10 mL) was added and stirred at room temperature for 1 hour, and saturated sodium bicarbonate (50 mL) was added to quench the reaction. Extract with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purified by preparative high performance liquid chromatography to obtain 1-((5-hydroxy-5-(trifluoromethyl)tetrahydro- 2H -pyran-2-yl)methyl)-3,7-dimethyl -1 H - purine -2,6- (3 H, 7 H) - dione (15.0 mg, yellow solid), yield: 30%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 8.28 (s, 1H), 4.39-4.10 (m, 2H), 4.05 (s, 3H), 3.93-3.89 (m, 2H), 3.55 (s, 3H), 3.32-3.27 (m, 1H), 1.89-1.65 (m, 4H). MS-ESI calculated value [M+H] + 363, measured value 363.

實施例25。 Example 25.

1-(4-(3-羥基戊烷-3-基)苄基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1- (4- (3-hydroxy-pentan-3-yl) benzyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

Figure 105135042-A0305-02-0090-90
Figure 105135042-A0305-02-0090-90

第一步。 first step.

4-((3，7-二甲基-2，6-氧代-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)苯甲酸甲酯。 Methyl 4-((3,7-dimethyl-2,6-oxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)methyl)benzoate.

氮氣保護，在25℃和下將3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(180mg，1.00mmol)，4-(溴甲基)苯甲酸甲酯(251mg，1.10mmol)， 碘化鉀(55.0mg，0.33mmol)和碳酸鉀(179mg，1.30mmol)溶解於無水N，N-二甲基甲醯胺(4mL)中，並加熱到110℃，攪拌3小時。冷却至25℃後，加水稀釋，用乙酸乙酯萃取(30mL x 2)。合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮用色譜矽膠柱純化(1：1石油醚/乙酸乙酯，Rf=0.3)得到4-((3，7-二甲基-2，6-氧代-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)苯甲酸甲酯(300mg，白色固體)，產率：91%。MS-ESI計算值[M+H]+ 329，實測值329。 Nitrogen, at 25 deg.] C and a 3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (180mg, 1.00mmol), 4- (bromomethyl) benzene Methyl formate (251mg, 1.10mmol), potassium iodide (55.0mg, 0.33mmol) and potassium carbonate (179mg, 1.30mmol) were dissolved in anhydrous N , N -dimethylformamide (4mL) and heated to 110°C , Stir for 3 hours. After cooling to 25°C, it was diluted with water and extracted with ethyl acetate (30 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by chromatography on a silica gel column (1:1 petroleum ether/ethyl acetate, Rf=0.3) to obtain 4-((3,7-dimethyl-2) ,6-oxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)methyl)benzoic acid methyl ester (300mg, white solid), yield: 91%. MS-ESI calculated value [M+H] + 329, measured value 329.

第二步。 The second step.

1-(4-(3-羥基戊烷-3-基)苄基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1- (4- (3-hydroxy-pentan-3-yl) benzyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

將4-((3，7-二甲基-2，6-氧代-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)苯甲酸甲酯(200mg，0.610mmol)溶解於無水四氫呋喃(3mL)中。氮氣保護，-78℃下滴加乙基溴化鎂(3M乙醚溶液，1.2mL，3.60mmol)。反應液此溫度下攪拌0.5小時，自然升至25℃繼續反應1小時。加入飽和氯化銨水溶液淬滅(5mL)，用乙酸乙酯萃取(30mL x 2)。合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮用製備TLC板純化(1：3石油醚/乙酸乙酯，Rf=0.3)得到1-(4-(3-羥基戊烷-3-基)苄基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(190mg，白色固體)，產率：87%。1H NMR：(400MHz，Methonal-d 4 )δ7.85(s，1H)，7.32(d，J=8.0Hz，2H)，7.28(d，J=8.0Hz，2H)，5.25(s，2H)，3.96(s，3H)，3.52(s，3H)，1.82-1.72(m，4H)，0.69(t，J=7.2Hz，6H)。MS-ESI計算值[M+H]+ 357，實測值357。 Methyl 4-((3,7-dimethyl-2,6-oxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)methyl)benzoate (200mg, 0.610 mmol) was dissolved in dry tetrahydrofuran (3 mL). Under nitrogen protection, ethyl magnesium bromide (3M ether solution, 1.2 mL, 3.60 mmol) was added dropwise at -78°C. The reaction solution was stirred at this temperature for 0.5 hours, and naturally raised to 25°C to continue the reaction for 1 hour. It was quenched by adding saturated aqueous ammonium chloride solution (5 mL), and extracted with ethyl acetate (30 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified with a preparative TLC plate (1:3 petroleum ether/ethyl acetate, Rf=0.3) to obtain 1-(4-(3-hydroxypentane-3) - yl) benzyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (190 mg of, white solid), yield: 87%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.85 (s, 1H), 7.32 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 5.25 (s, 2H) ), 3.96 (s, 3H), 3.52 (s, 3H), 1.82-1.72 (m, 4H), 0.69 (t, J = 7.2 Hz, 6H). MS-ESI calculated value [M+H] + 357, measured value 357.

實施例26。 Example 26.

3，7-二甲基-1-(3-(1，1，1-三氟-2-羥基丙烷-2-基)苄基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3,7-Dimethyl-1-(3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzyl)-1 H -purine-2,6-(3 H , 7 H )-Diketone.

Figure 105135042-A0305-02-0092-91
Figure 105135042-A0305-02-0092-91

第一步。 first step.

3-乙醯基苯甲酸乙酯。 Ethyl 3-acetylbenzoate.

將3-乙醯基苯甲酸(500mg，3.05mmol)溶於N，N-二甲基甲醯胺(20mL)中，室溫條件下加入碘乙烷(475mg，3.05mmol)和碳酸鉀(632mg，4.57mmol)，室溫攪拌2小時後，反應液濃縮，加入乙酸乙酯(30mL)稀釋，有機相用水(20mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱層析法分離純化(5：1石油醚/乙酸乙酯，Rf=0.5)得到3-乙醯基苯甲酸乙酯(530mg，白色固體)，產率：90%。1H NMR：(400MHz，CDCl3)δ8.60(s，1H)，8.24(d，J=7.6Hz，1H)，8.15(d，J=7.6Hz，1H)，7.56(t，J=7.6Hz，1H)，4.41(q，J=7.2Hz，2H)，2.66(s，3H)，1.42(t，J=7.2Hz，3H)。MS-ESI計算值[M+H]+ 193，實測值193。 3-Acetylbenzoic acid (500mg, 3.05mmol) was dissolved in N , N -dimethylformamide (20mL), and iodoethane (475mg, 3.05mmol) and potassium carbonate (632mg) were added at room temperature. , 4.57mmol), after stirring for 2 hours at room temperature, the reaction solution was concentrated, diluted with ethyl acetate (30mL), the organic phase was washed with water (20mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure using a silica gel column Separation and purification by chromatography (5:1 petroleum ether/ethyl acetate, Rf=0.5) gave ethyl 3-acetylbenzoate (530 mg, white solid), yield: 90%. 1 H NMR: (400MHz, CDCl 3 ) δ8.60 (s, 1H), 8.24 (d, J = 7.6 Hz, 1H), 8.15 (d, J = 7.6 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 4.41 (q, J = 7.2 Hz, 2H), 2.66 (s, 3H), 1.42 (t, J = 7.2 Hz, 3H). MS-ESI calculated value [M+H] + 193, measured value 193.

第二步。 The second step.

乙基3-(1，1，1-三氟-2-羥基丙烷-2-基)苯甲酸乙酯。 Ethyl ethyl 3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzoate.

將3-乙醯基苯甲酸乙酯(500mg，2.60mmol)溶於四氫呋喃(20mL)中，室溫條件下加入三氟甲基三甲基矽烷(370mg，2.60mmol)和氟化銫(79.0mg，0.520mmol)。室溫攪拌12小時，反應液加入乙酸乙酯(30mL)稀釋，有機相用水(20mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱層析法分離純化(2：1石油醚/乙酸乙酯，Rf=0.5)得到乙基3-(1，1，1-三氟-2-羥基丙烷-2-基)苯甲酸乙酯(600mg，黃色固體)，產率：88%。1H NMR：(400MHz，CDCl3)δ8.26(s，1H)，8.05(d，J=7.6Hz，1H)，7.80(d，J=7.6Hz，1H)，7.48(t，J=7.6Hz，1H)，4.39(q，J=7.2Hz，2H)，1.82(s，3H)，1.40(t，J=7.2Hz，3H)。MS-ESI計算值[M+H]+ 263，實測值263。 Ethyl 3-acetylbenzoate (500mg, 2.60mmol) was dissolved in tetrahydrofuran (20mL), and trifluoromethyltrimethylsilane (370mg, 2.60mmol) and cesium fluoride (79.0mg) were added at room temperature. , 0.520mmol). After stirring for 12 hours at room temperature, the reaction solution was diluted with ethyl acetate (30 mL), the organic phase was washed with water (20 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and separated and purified by silica gel column chromatography (2: 1 petroleum ether/ethyl acetate, Rf=0.5) to obtain ethyl 3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzoate (600mg, yellow solid), yield: 88%. 1 H NMR: (400MHz, CDCl 3 ) δ 8.26 (s, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 1.82 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H). MS-ESI calculated value [M+H] + 263, measured value 263.

第三步。 third step.

1，1，1-三氟-2-(3-羥基甲基)苯基)丙-2-醇。 1,1,1-Trifluoro-2-(3-hydroxymethyl)phenyl)propan-2-ol.

將乙基3-(1，1，1-三氟-2-羥基丙烷-2-基)苯甲酸乙酯(500mg，1.91mmol)溶於四氫呋喃(20mL)中，反應液於0℃條件下加入氫化鋰鋁(108mg，2.87mmol)，室溫攪拌2小時，反應液中分別加入水(0.1mL)，15%氫氧化鈉(0.1mL)和水(0.3mL)，攪拌20分鐘。反應液中加入乙酸乙酯(30mL)稀釋，有機相用水(20mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到1，1，1-三氟-2-(3-羥基甲基)苯基)丙-2-醇(400mg，黃色固體)，產率：95%。 Ethyl ethyl 3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzoate (500mg, 1.91mmol) was dissolved in tetrahydrofuran (20mL), and the reaction solution was added at 0°C Lithium aluminum hydride (108 mg, 2.87 mmol) was stirred at room temperature for 2 hours. Water (0.1 mL), 15% sodium hydroxide (0.1 mL) and water (0.3 mL) were added to the reaction solution, and the mixture was stirred for 20 minutes. The reaction solution was diluted with ethyl acetate (30 mL), the organic phase was washed with water (20 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 1,1,1-trifluoro-2-(3-hydroxymethyl) (Yl)phenyl)propan-2-ol (400 mg, yellow solid), yield: 95%.

1H NMR：(400MHz，CDCl3)δ7.62(s，1H)，8.05(d，J=7.6Hz，1H)，7.41-7.37(m，2H)，4.73(s，2H)，1.80(s，3H)。MS-ESI計算值[M+H]+ 221，實測值221。 1 H NMR: (400MHz, CDCl 3 ) δ 7.62 (s, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.41-7.37 (m, 2H), 4.73 (s, 2H), 1.80 (s , 3H). MS-ESI calculated value [M+H] + 221, measured value 221.

第四步。 the fourth step.

3-(1，1，1-三氟-2-羥基丙烷-2-基)苄基甲磺酸酯。 3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzyl methanesulfonate.

將1,1,1-三氟-2-(3-羥基甲基)苯基)丙-2-醇(400mg，1.82mmol)和三乙胺(275mg，2.72mmol)溶於二氯甲烷(20mL)中，反應液於0℃條件下加入甲烷磺醯氯(250mg，2.18mmol)，攪拌2小時，加入二氯甲烷(30mL)稀釋，有機相用水(20mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到3-(1，1，1-三氟-2-羥基丙烷-2-基)苄基甲磺酸酯(500mg，黃色油狀物)，產率：92%。 Dissolve 1,1,1-trifluoro-2-(3-hydroxymethyl)phenyl)propan-2-ol (400mg, 1.82mmol) and triethylamine (275mg, 2.72mmol) in dichloromethane (20mL ), add methanesulfonyl chloride (250mg, 2.18mmol) to the reaction solution at 0°C, stir for 2 hours, add dichloromethane (30mL) to dilute, wash the organic phase with water (20mL x 2), dry with anhydrous sodium sulfate, After filtration, the filtrate was concentrated under reduced pressure to obtain 3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzyl methanesulfonate (500 mg, yellow oil), yield: 92%.

MS-ESI計算值[M+H]+ 299，實測值299。 MS-ESI calculated value [M+H] + 299, measured value 299.

第五步。 the fifth step.

3，7-二甲基-1-(3-(1，1，1-三氟-2-羥基丙烷-2-基)苄基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3,7-Dimethyl-1-(3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzyl)-1 H -purine-2,6-(3 H , 7 H )-Diketone.

將3-(1，1，1-三氟-2-羥基丙烷-2-基)苄基甲磺酸酯(100mg，0.335mmol)和3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(60.0mg，0.335mmol)溶於N，N-二甲基甲醯胺(20mL)中，室溫條件下加入碳酸鉀(70.0mg，0.502mmol)和碘化鉀(6.00mg，0.0335mmol)，加熱100℃攪拌2小時後，反應液冷却濃縮，加入乙酸乙酯(30mL)稀釋，有機相用水(20mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用高效液相色譜純化得到3，7-二甲基-1-(3-(1，1，1-三氟-2-羥基丙烷-2-基)苄基)-1H-嘌呤-2，6-(3H，7H)-二酮(30.0mg，白色固體)，產率：23%。1H NMR：(400MHz，CDCl3)δ7.90(s，1H)，7.70(s，1H)，7.49(d，J=7.6Hz，1H)，7.38-7.32(m，2H)，5.21(s，2H)，4.00(s，3H)，3.55(s，3H)，1.71(s，3H)。MS-ESI計算值[M+H]+ 383，實測值383。 Combine 3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzyl methanesulfonate (100mg, 0.335mmol) and 3,7-dimethyl- 1H -purine-2, 6-(3 H , 7 H )-dione (60.0mg, 0.335mmol) was dissolved in N,N -dimethylformamide (20mL), potassium carbonate (70.0mg, 0.502mmol) was added at room temperature And potassium iodide (6.00mg, 0.0335mmol), heated at 100°C and stirred for 2 hours, the reaction solution was cooled and concentrated, diluted with ethyl acetate (30mL), the organic phase was washed with water (20mL x 2), dried with anhydrous sodium sulfate, filtered, and the filtrate Concentrate under reduced pressure. Purified by high performance liquid chromatography to obtain 3,7-dimethyl-1-(3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)benzyl)-1 H -purine-2, 6-(3 H , 7 H )-dione (30.0 mg, white solid), yield: 23%. 1 H NMR: (400MHz, CDCl 3 ) δ 7.90 (s, 1H), 7.70 (s, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.38-7.32 (m, 2H), 5.21 (s , 2H), 4.00 (s, 3H), 3.55 (s, 3H), 1.71 (s, 3H). MS-ESI calculated value [M+H] + 383, measured value 383.

實施例27。 Example 27.

3，7-二甲基-1-(4-(1，1，1-三氟-2-羥基丙基-2-基)苯基)-1H-嘌呤-2，6(3H，7H)-二酮。 3,7-Dimethyl-1-(4-(1,1,1-trifluoro-2-hydroxypropyl-2-yl)phenyl)-1 H -purine-2,6(3 H , 7 H )-Diketone.

Figure 105135042-A0305-02-0095-92
Figure 105135042-A0305-02-0095-92

第一步。 first step.

4-(1，1，1-三氟-2-羥基丙基-2-基)苯甲酸甲酯。 Methyl 4-(1,1,1-trifluoro-2-hydroxypropyl-2-yl)benzoate.

氮氣保護，0℃將4-乙醯基苯甲酸甲酯(10.0g，56.1mmol)和三甲基(三氟甲基)矽烷(16.0g，112mmol)溶於無水四氫呋喃(150mL)中，並慢慢滴加四丁基氟化銨(22.0g，84.2mmol)。反應緩慢升至室溫，攪拌過夜。加入水(50mL)淬滅反應。用乙酸乙酯萃取(50mL x 3)。合併有機相，依次用飽和碳酸氫鈉水溶液，飽和食鹽水洗滌，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到產物4-(1，1，1-三氟-2-羥基丙基-2-基)苯甲酸甲酯(7.00g，黃色液體)，產率：50%。1H NMR：(400MHz，CDCl3)δ8.04(d，J=8.0Hz，2H)，7.68(d，J=8.0Hz，2H)，3.92(s，3H)，3.27(s，1H)，1.80(s，3H)。MS-ESI計算值[M+H]+ 249，實測值249。 Under nitrogen protection, dissolve methyl 4-acetylbenzoate (10.0g, 56.1mmol) and trimethyl(trifluoromethyl)silane (16.0g, 112mmol) in anhydrous tetrahydrofuran (150mL) at 0°C and slowly Slowly add tetrabutylammonium fluoride (22.0 g, 84.2 mmol) dropwise. The reaction was slowly raised to room temperature and stirred overnight. The reaction was quenched by adding water (50 mL). Extract with ethyl acetate (50 mL x 3). The organic phases were combined, washed successively with saturated aqueous sodium bicarbonate solution, saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the product 4-(1,1,1-trifluoro-2-hydroxypropyl-2) -Methyl)benzoate (7.00 g, yellow liquid), yield: 50%. 1 H NMR: (400MHz, CDCl 3 ) δ 8.04 (d, J = 8.0 Hz, 2H), 7.68 (d, J = 8.0 Hz, 2H), 3.92 (s, 3H), 3.27 (s, 1H), 1.80(s, 3H). MS-ESI calculated value [M+H] + 249, measured value 249.

第二步。 The second step.

1，1，1-三氟-2-(4-(羥基甲基)苯基)丙基-2-醇。 1,1,1-Trifluoro-2-(4-(hydroxymethyl)phenyl)propyl-2-ol.

在氮氣保護，0℃將氫化鋁鋰(1.61g，42.3mmol)緩慢加入甲基4-(1，1，1-三氟-2-羥基丙基-2-基)苯甲酸甲酯(7.00g，28.2mmol)的四氫呋喃(150mL)溶液中。反應液在0℃攪拌3小時。0℃下，依次緩慢加 入水(1.60mL)，15%氫氧化鈉溶液(1.60mL)及水(4.80mL)。過濾，濾液減壓濃縮得到產物1，1，1-三氟-2-(4-(羥基甲基)苯基)丙基-2-醇(2.40g，黃色液體)，產率：93%。1H NMR：(400MHz，CDCl3)δ7.55(d，J=8.0Hz，2H)，7.34(d，J=8.0Hz，2H)，4.66(s，2H)，3.37(s，1H)，2.39(s，1H)，1.75(s，3H)。MS-ESI計算值[M+H]+ 221，實測值221。 Under nitrogen protection, lithium aluminum hydride (1.61g, 42.3mmol) was slowly added to methyl 4-(1,1,1-trifluoro-2-hydroxypropyl-2-yl)benzoate (7.00g) at 0°C , 28.2mmol) in tetrahydrofuran (150mL) solution. The reaction solution was stirred at 0°C for 3 hours. At 0°C, slowly add water (1.60 mL), 15% sodium hydroxide solution (1.60 mL) and water (4.80 mL) in sequence. After filtration, the filtrate was concentrated under reduced pressure to obtain the product 1,1,1-trifluoro-2-(4-(hydroxymethyl)phenyl)propyl-2-ol (2.40 g, yellow liquid), yield: 93%. 1 H NMR: (400MHz, CDCl 3 ) δ 7.55 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 4.66 (s, 2H), 3.37 (s, 1H), 2.39 (s, 1H), 1.75 (s, 3H). MS-ESI calculated value [M+H] + 221, measured value 221.

第三步。 third step.

4-(1，1，1-三氟-2-羥基丙基-2-基)苯基甲磺酸酯。 4-(1,1,1-trifluoro-2-hydroxypropyl-2-yl)phenyl methanesulfonate.

將1，1，1-三氟-2-(4-(羥基甲基)苯基)丙基-2-醇(5.80g，26.3mmol)及二異丙基乙基胺(10.2g，79.0mmol)溶於二氯甲烷(80mL)中，在0℃下緩慢加入甲烷磺醯氯(4.53g，39.5mmol)。反應液於0℃下，攪拌0.5小時。加入飽和氯化銨水溶液(50mL)淬滅反應，用二氯甲烷萃取(20mL x 3)。合併有機相，用飽和碳酸氫鈉水溶液(50mL)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法純化(5：1石油醚/乙酸乙酯，Rf=0.4)得到產物4-(1，1，1-三氟-2-羥基丙基-2-基)苯基甲磺酸酯(3.45g，黃色油狀物)，產率：44%。1H NMR：(400MHz，CDCl3)δ7.66(d，J=8.0Hz，2H)，7.46(d，J=8.0Hz，2H)，5.26(s，2H)，2.96(s，3H)，2.84(s，1H)，1.80(s，3H)。MS-ESI計算值[M+H]+ 299，實測值299。 Combine 1,1,1-trifluoro-2-(4-(hydroxymethyl)phenyl)propyl-2-ol (5.80g, 26.3mmol) and diisopropylethylamine (10.2g, 79.0mmol) ) Was dissolved in dichloromethane (80 mL), and methanesulfonyl chloride (4.53 g, 39.5 mmol) was slowly added at 0°C. The reaction solution was stirred at 0°C for 0.5 hour. The reaction was quenched by adding saturated aqueous ammonium chloride solution (50 mL), and extracted with dichloromethane (20 mL x 3). The organic phases were combined, washed with saturated aqueous sodium bicarbonate solution (50 mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified by silica gel column chromatography (5:1 petroleum ether/ethyl acetate, Rf=0.4) to obtain the product 4-(1,1,1-trifluoro-2-hydroxypropyl-2-yl)phenyl methanesulfonate (3.45 g, yellow oil), yield: 44%. 1 H NMR: (400MHz, CDCl 3 ) δ 7.66 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 5.26 (s, 2H), 2.96 (s, 3H), 2.84 (s, 1H), 1.80 (s, 3H). MS-ESI calculated value [M+H] + 299, measured value 299.

第四步。 the fourth step.

3，7-二甲基-1-(4-(1，1，1-三氟-2-羥基丙基-2-基)苯基)-1H-嘌呤-2，6(3H，7H)-二酮。 3,7-Dimethyl-1-(4-(1,1,1-trifluoro-2-hydroxypropyl-2-yl)phenyl)-1 H -purine-2,6(3 H , 7 H )-Diketone.

將4-(1，1，1-三氟-2-羥基丙基-2-基)苯基甲磺酸酯(1.95g，10.8mmol)，3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(652mg，3.62mmol) 及碳酸鉀(2.99g，21.6mmol)和碘化鉀(180mg，1.08mmol)溶於N，N-二甲基甲醯胺(30mL)。反應液加熱至130℃，攪拌3小時。反應液冷却至室溫，加入飽和食鹽水(20mL)，用乙酸乙酯萃取(100mL x 3)。合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法分離純化(1：2石油醚/乙酸乙酯，Rf=0.3)得到產物3，7-二甲基-1-(4-(1，1，1-三氟-2-羥基丙基-2-基)苯基)-1H-嘌呤-2，6(3H，7H)-二酮(1.27g，白色固體)，產率：31%。1H NMR：(400MHz，CDCl3)δ7.57-7.55(m，5H)，5.20(s，2H)，3.99(s，3H)，3.58(s，3H)，2.60(s，1H)，1.74(s，3H)。MS-ESI計算值[M+H]+ 383，實測值383。 Add 4-(1,1,1-trifluoro-2-hydroxypropyl-2-yl)phenyl methanesulfonate (1.95g, 10.8mmol), 3,7-dimethyl- 1H -purine- 2,6 (3 H, 7 H) - dione (652mg, 3.62mmol) and potassium carbonate (2.99g, 21.6mmol) and potassium iodide (180mg, 1.08mmol) was dissolved in N, N - dimethylformamide ( 30mL). The reaction solution was heated to 130°C and stirred for 3 hours. The reaction solution was cooled to room temperature, saturated brine (20 mL) was added, and extracted with ethyl acetate (100 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and separated and purified by silica gel column chromatography (1:2 petroleum ether/ethyl acetate, Rf=0.3) to obtain the product 3,7-dimethyl-1 -(4-(1,1,1-Trifluoro-2-hydroxypropyl-2-yl)phenyl)-1 H -purine-2,6(3 H ,7 H )-dione (1.27g, White solid), yield: 31%. 1 H NMR: (400MHz, CDCl 3 ) δ 7.57-7.55 (m, 5H), 5.20 (s, 2H), 3.99 (s, 3H), 3.58 (s, 3H), 2.60 (s, 1H), 1.74 (s, 3H). MS-ESI calculated value [M+H] + 383, measured value 383.

實施例28。 Example 28.

Figure 105135042-A0305-02-0097-93
Figure 105135042-A0305-02-0097-93

第一步。 first step.

6-溴烟酸甲酯。 Methyl 6-bromonicotinate.

將6-溴烟酸(1.00g，4.95mmol)溶於N，N二甲基甲醯胺(30mL)中，加入碘甲烷(0.703g，4.95mmol)和碳酸鉀(1.03g，7.43mmol)。 反應液於20℃攪拌12小時。反應液加入水(100mL)稀釋，用乙酸乙酯萃取(30mL x 3)，有機相用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱層析法分離純化(2：1石油醚/乙酸乙酯，Rf=0.5)得到6-溴烟酸甲酯(1.00g，白色固體)，產率：94%。MS-ESI計算值[M+H]+ 216和218，實測值216和218。 6-Bromonicotinic acid (1.00 g, 4.95 mmol) was dissolved in N , N dimethylformamide (30 mL), and methyl iodide (0.703 g, 4.95 mmol) and potassium carbonate (1.03 g, 7.43 mmol) were added. The reaction solution was stirred at 20°C for 12 hours. The reaction solution was diluted with water (100mL), extracted with ethyl acetate (30mL x 3), the organic phase was dried with anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and separated and purified by silica gel column chromatography (2:1 petroleum ether/ Ethyl acetate, Rf=0.5) to obtain methyl 6-bromonicotinate (1.00 g, white solid), yield: 94%. MS-ESI calculated values [M+H] + 216 and 218, and measured values 216 and 218.

第二步。 The second step.

(6-溴吡啶-3-基)甲醇。 (6-Bromopyridin-3-yl)methanol.

將6-溴烟酸甲酯(1.00g，4.63mmol)溶於四氫呋喃(20mL)中，0℃下，加入四氫鋰鋁(351mg，9.26mmol)，反應1小時。加入水(10mL)淬滅反應。用乙酸乙酯萃取(20mL x 3)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用用矽膠柱色譜法分離純化(1：1石油醚/乙酸乙酯，Rf=0.3)得到(6-溴吡啶-3-基)甲醇(600mg，黃色油狀物)，產率：69%。MS-ESI計算值[M+H]+ 188和190，實測值188和190。 Methyl 6-bromonicotinate (1.00 g, 4.63 mmol) was dissolved in tetrahydrofuran (20 mL), at 0° C., lithium aluminum tetrahydrogen (351 mg, 9.26 mmol) was added, and the reaction was carried out for 1 hour. The reaction was quenched by adding water (10 mL). It was extracted with ethyl acetate (20mL x 3), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and separated and purified by silica gel column chromatography (1:1 petroleum ether/ethyl acetate, Rf=0.3) to obtain (6- Bromopyridin-3-yl)methanol (600 mg, yellow oil), yield: 69%. MS-ESI calculated values [M+H] + 188 and 190, and measured values 188 and 190.

第三步。 third step.

(6-溴吡啶-3-基)甲基甲磺酸酯。 (6-Bromopyridin-3-yl)methanesulfonate.

將(6-溴吡啶-3-基)甲醇(1.00g，5.32mmol)和三乙胺(1.18g，11.6mmol)溶於二氯甲烷(20mL)中，0℃條件下加入甲磺醯氯(1.38g，12.0mmol)。反應液於室溫攪拌2小時後，加入二氯甲烷(20mL)稀釋，用飽和碳酸氫鈉(30mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱層析法分離純化(4：1石油醚/乙酸乙酯，Rf=0.5)得到(6-溴吡啶-3-基)甲基甲磺酸酯(1.20g，無色油狀物)，產率：85%。MS-ESI計算值[M+H]+ 266和268，實測值266和268。 (6-Bromopyridin-3-yl)methanol (1.00g, 5.32mmol) and triethylamine (1.18g, 11.6mmol) were dissolved in dichloromethane (20mL), and methanesulfonyl chloride ( 1.38g, 12.0mmol). After the reaction solution was stirred at room temperature for 2 hours, it was diluted with dichloromethane (20mL), washed with saturated sodium bicarbonate (30mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography. Purification (4:1 petroleum ether/ethyl acetate, Rf=0.5) gave (6-bromopyridin-3-yl)methanesulfonate (1.20 g, colorless oil), yield: 85%. MS-ESI calculated values [M+H] + 266 and 268, measured values 266 and 268.

第四步。 the fourth step.

1-((6-溴吡啶-3-基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 L - ((6-bromo-3-yl) methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

將(6-溴吡啶-3-基)甲基甲磺酸酯(500mg，1.88mmol)溶於N，N-二甲基甲醯胺(20mL)中，反應液於室溫條件下加入3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(338mg，1.88mmol)，碳酸鉀(389mg，2.82mmol)和碘化鉀(184mg，1.11mmol)。反應液加熱至100℃，反應2小時，加入乙酸乙酯(20mL)稀釋，有機相用飽和碳酸氫鈉(20mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱層析法分離純化(1：1石油醚/乙酸乙酯，Rf=0.3)得到1-((6-溴吡啶-3-基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(300mg，黃色固體)，產率：46%。MS-ESI計算值[M+H]+ 350和352，實測值350和352。 (6-Bromopyridin-3-yl)methanesulfonate (500mg, 1.88mmol) was dissolved in N , N -dimethylformamide (20mL), the reaction solution was added 3 at room temperature, 7-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (338mg, 1.88mmol), potassium carbonate (389mg, 2.82mmol) and potassium iodide (184mg, 1.11mmol). The reaction solution was heated to 100°C, reacted for 2 hours, and diluted with ethyl acetate (20mL). The organic phase was washed with saturated sodium bicarbonate (20mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Separation and purification by analysis (1:1 petroleum ether/ethyl acetate, Rf=0.3) to obtain 1-((6-bromopyridin-3-yl)methyl)-3,7-dimethyl-1 H -purine- 2,6( 3H , 7H )-dione (300mg, yellow solid), yield: 46%. MS-ESI calculated values [M+H] + 350 and 352, and measured values 350 and 352.

第五步。 the fifth step.

1-((6-乙醯基吡啶-3-基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 L - ((6- acetyl-3-yl) methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

將1-((6-溴吡啶-3-基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(2.00g，5.71mmol)溶於1，4-二氧六環(50mL)中，反應液於室溫條件下加入三丁基(1-乙氧基乙烯基)錫烷(8.25g，22.8mmol)和四三苯基膦鈀(329mg，0.285mmol)。反應液加熱至120℃攪拌2小時，反應液冷却至室溫，加入乙酸乙酯(70mL)稀釋，用飽和碳酸氫鈉(20mL)洗滌(30mL x 2)，無水硫酸鈉乾燥，過濾，濃縮，用矽膠柱層析法分離純化(3：1石油醚/乙酸乙酯，Rf=0.3)得到1-((6-乙醯基吡啶-3-基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(1.00g，黃色固體)，產率：56%。1H NMR：(400MHz，CDCl3)δ8.83(s，1H)，8.00-7.98(m，1H)，7.95-7.93(m，1H)，7.54(s，1H)，5.27(s，2H)，4.01(s， 3H)，3.59(s，3H)，2.71(s，3H)。MS-ESI計算值[M+H]+ 314，實測值314。 1 - ((6-bromo-3-yl) methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (2.00g, 5.71mmol ) Was dissolved in 1,4-dioxane (50mL), and the reaction mixture was added with tributyl(1-ethoxyvinyl)stannane (8.25g, 22.8mmol) and tetratriphenyl at room temperature. Phosphine palladium (329 mg, 0.285 mmol). The reaction solution was heated to 120°C and stirred for 2 hours. The reaction solution was cooled to room temperature, diluted with ethyl acetate (70mL), washed with saturated sodium bicarbonate (20mL) (30mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated. Separated and purified by silica gel column chromatography (3:1 petroleum ether/ethyl acetate, Rf=0.3) to obtain 1-((6-acetylpyridin-3-yl)methyl)-3,7-dimethyl -1 H -purine-2,6(3 H , 7 H )-dione (1.00 g, yellow solid), yield: 56%. 1 H NMR: (400MHz, CDCl 3 ) δ 8.83 (s, 1H), 8.00-7.98 (m, 1H), 7.95-7.93 (m, 1H), 7.54 (s, 1H), 5.27 (s, 2H) , 4.01 (s, 3H), 3.59 (s, 3H), 2.71 (s, 3H). MS-ESI calculated value [M+H] + 314, measured value 314.

第六步。 The sixth step.

3，7-二甲基-1-((6-(1，1，1-三氟-2-羥基丙烷-2-基)吡啶-3-基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮。 3,7-Dimethyl-1-((6-(1,1,1-trifluoro-2-hydroxypropan-2-yl)pyridin-3-yl)methyl)-1 H -purine-2, 6(3 H , 7 H )-dione.

將1-((6-乙醯基吡啶-3-基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(150mg，0.478mmol)溶於四氫呋喃(30mL)中，室溫條件下加入三氟甲基三甲基矽烷(102mg，0.718mmol)和氟化銫(73.0mg，0.478mmol)。反應液在室溫攪拌12小時，加入四丁基氟化銨(50.0mg，0.207mmol)，室溫攪拌30分鐘後加入乙酸乙酯(20mL)稀釋，有機相用飽和碳酸氫鈉(20mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮用高效液相色譜純化得3，7-二甲基-1-((6-(1，1，1-三氟-2-羥基丙烷-2-基)吡啶-3-基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮(50mg，白色固體)，產率：27%。1H NMR：(400MHz，CDCl3)δ8.76(s，1H)，7.99(d，J=8.0Hz，1H)，7.53(s，1H)，7.44(d，J=8.0Hz，1H)，5.23(s，2H)，3.99(s，3H)，3.58(s，3H)，1.68(s，3H)。MS-ESI計算值[M+H]+ 384，實測值384。 1 - ((6-acetyl-3-yl) methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (150mg, 0.478 mmol) was dissolved in tetrahydrofuran (30 mL), and trifluoromethyltrimethylsilane (102 mg, 0.718 mmol) and cesium fluoride (73.0 mg, 0.478 mmol) were added at room temperature. The reaction solution was stirred at room temperature for 12 hours, tetrabutylammonium fluoride (50.0mg, 0.207mmol) was added, stirred at room temperature for 30 minutes and then diluted with ethyl acetate (20mL). The organic phase was diluted with saturated sodium bicarbonate (20mL x 2 ) Was washed, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by high performance liquid chromatography to obtain 3,7-dimethyl-1-((6-(1,1,1-trifluoro-2-hydroxypropane- 2- yl) pyridin-3-yl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (50mg, white solid), yield: 27%. 1 H NMR: (400MHz, CDCl 3 )δ8.76 (s, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.53 (s, 1H), 7.44 (d, J = 8.0 Hz, 1H), 5.23 (s, 2H), 3.99 (s, 3H), 3.58 (s, 3H), 1.68 (s, 3H). MS-ESI calculated value [M+H] + 384, measured value 384.

實施例29。 Example 29.

3，7-二甲基-1-[[5-(2，2，2-三氟-1-羥基-1-甲基-乙基)-2-吡啶基]甲基]嘌呤-2，6-二酮。 3,7-Dimethyl-1-[[5-(2,2,2-Trifluoro-1-hydroxy-1-methyl-ethyl)-2-pyridyl]methyl]purine-2,6 -Diketone.

Figure 105135042-A0305-02-0100-94
Figure 105135042-A0305-02-0100-94
Figure 105135042-A0305-02-0101-97
Figure 105135042-A0305-02-0101-97

第一步。 first step.

1-[6-(溴甲基)-3-吡啶基]乙酮。 1-[6-(Bromomethyl)-3-pyridyl]ethanone.

將1-(6-甲基-3-吡啶基)乙酮(500mg，3.70mmol)，N-溴代丁二醯亞胺(658mg，3.70mmol)，偶氮二異丁腈(182mg，1.11mmol)溶於四氯化碳(20mL)中，90℃反應12小時。加入飽和硫代硫酸鈉溶液(30mL)淬滅反應。用二氯甲烷萃取(10mL x 3)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到1-[6-(溴甲基)-3-吡啶基]乙酮(125mg，黃色油狀)，產率：16%。MS-ESI計算值[M+H]+ 214，216，實測值214，216。 Combine 1-(6-methyl-3-pyridyl)ethanone (500mg, 3.70mmol), N -bromosuccinimide (658mg, 3.70mmol), azobisisobutyronitrile (182mg, 1.11mmol) ) Was dissolved in carbon tetrachloride (20mL) and reacted at 90°C for 12 hours. The reaction was quenched by adding saturated sodium thiosulfate solution (30 mL). Extract with dichloromethane (10mL x 3), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain 1-[6-(bromomethyl)-3-pyridyl]ethanone (125mg, yellow oil), Yield: 16%. MS-ESI calculated value [M+H] + 214,216, measured value 214,216.

第二步。 The second step.

1-[(5-乙醯基-2-吡啶基)甲基]-3，7-二甲基嘌呤-2，6-二酮。 1-[(5-Acetyl-2-pyridyl)methyl]-3,7-dimethylpurine-2,6-dione.

將1-[6-(溴甲基)-3-吡啶基]乙酮(100mg，0.467mmol)，3，7-二甲基嘌呤-2，6-二酮(84.2mg，0.467mmol)，碘化鉀(7.70mg，0.0467mmol)和碳酸鉀(194mg，1.40mmol)溶於N，N-二甲基甲醯胺(10mL)中。反應液升溫至120℃，攪拌3小時。冷却至室溫，過濾，濾液減壓濃縮用製備TLC板分離純化(乙酸乙酯，Rf值=0.3)得到1-[(5-乙醯基-2-吡啶基)甲基]-3，7-二甲基嘌呤-2，6-二酮(50.0mg，黃色固體)，產率：34%。MS-ESI計算值[M+H]+ 314，實測值314。 Combine 1-[6-(bromomethyl)-3-pyridyl]ethanone (100mg, 0.467mmol), 3,7-dimethylpurine-2,6-dione (84.2mg, 0.467mmol), potassium iodide (7.70 mg, 0.0467 mmol) and potassium carbonate (194 mg, 1.40 mmol) were dissolved in N , N -dimethylformamide (10 mL). The reaction solution was heated to 120°C and stirred for 3 hours. Cool to room temperature, filter, and concentrate the filtrate under reduced pressure. Separate and purify with a preparative TLC plate (ethyl acetate, Rf value=0.3) to obtain 1-[(5-acetyl-2-pyridyl)methyl]-3,7 -Dimethylpurine-2,6-dione (50.0 mg, yellow solid), yield: 34%. MS-ESI calculated value [M+H] + 314, measured value 314.

第三步。 third step.

3，7-二甲基-1-[[5-(2，2，2-三氟-1-羥基-1-甲基-乙基)-2-吡啶基]甲基]嘌呤-2，6-二酮。 3,7-Dimethyl-1-[[5-(2,2,2-Trifluoro-1-hydroxy-1-methyl-ethyl)-2-pyridyl]methyl]purine-2,6 -Diketone.

將1-[(5-乙醯基-2-吡啶基)甲基]-3，7-二甲基嘌呤-2，6-二酮(50.0mg，0.159mmol)，氟化銫(24.2mg，0.159mmol)溶於四氫呋喃(10mL)中，室溫下加入三甲基-三氟甲基-矽烷(113mg，0.798mmol)，攪拌12小時。加入水(20mL)淬滅反應。用乙酸乙酯萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用製備型高效液相色譜純化，得到3，7-二甲基-1-[[5-(2，2，2-三氟-1-羥基-1-甲基-乙基)-2-吡啶基]甲基]嘌呤-2，6-二酮(10.0mg，黃色固體)，產率：16%。1H NMR：(400MHz，Methonal-d 4 )δ8.96(s，1H)，8.75(d，J=8.0Hz，1H)，8.06(d，J=8.0Hz，1H)，7.97(s，1H)，5.55(s，2H)，4.00(s，3H)，3.57(s，3H)，1.86(s，3H)。MS-ESI計算值[M+H]+ 384，實測值384。 1-[(5-Acetyl-2-pyridyl)methyl]-3,7-dimethylpurine-2,6-dione (50.0mg, 0.159mmol), cesium fluoride (24.2mg, 0.159 mmol) was dissolved in tetrahydrofuran (10 mL), trimethyl-trifluoromethyl-silane (113 mg, 0.798 mmol) was added at room temperature, and stirred for 12 hours. The reaction was quenched by adding water (20 mL). Extract with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purified by preparative high performance liquid chromatography to obtain 3,7-dimethyl-1-[[5-(2,2,2-trifluoro-1-hydroxy-1-methyl-ethyl)-2-pyridine Yl]methyl]purine-2,6-dione (10.0 mg, yellow solid), yield: 16%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 8.96 (s, 1H), 8.75 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.97 (s, 1H) ), 5.55 (s, 2H), 4.00 (s, 3H), 3.57 (s, 3H), 1.86 (s, 3H). MS-ESI calculated value [M+H] + 384, measured value 384.

實施例30。 Example 30.

3，7-二甲基-1-((5(1，1，1-三氟-2-羥基丙烷-2-基)吡嗪-2-基)甲基)-嘌呤-2，6(3H，7H)-二酮。 3,7-Dimethyl-1-((5(1,1,1-trifluoro-2-hydroxypropan-2-yl)pyrazin-2-yl)methyl)-purine-2,6(3 H , 7 H )-diketone.

Figure 105135042-A0305-02-0102-98
Figure 105135042-A0305-02-0102-98

第一步。 first step.

N-甲氧基-N，5-二甲基吡嗪-2-甲醯胺 N -Methoxy- N ,5-Dimethylpyrazine-2-carboxamide

將5-甲基吡嗪-2-羧酸(2.00g，14.5mmol)，1-羥基苯並三唑(391mg，2.90mmol)，1-(3-二甲氨基丙基)-3-乙基碳二亞胺鹽酸鹽(1.69g，17.4mmol)溶於無水二氯甲烷(10mL)和三氯甲烷(30mL)中，在氮氣保護，0℃時緩慢加入三乙胺(1.76g，17.4mmol)，反應液在25℃攪拌12小時。加入水(50mL)淬滅反應。反應液用乙酸乙酯(50mL x 3)萃取，合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，剩餘物用矽膠柱色譜法純化(20：1石油醚/乙酸乙酯，Rf=0.1)得到N-甲氧基-N，5-二甲基吡嗪-2-甲醯胺(2.00g，黃色油狀物)，產率：76%。1H NMR：(400MHz，CDCl3)δ8.80(s，1H)，8.45(s，1H)，3.73(s，3H)，3.40(s，3H)，2.61(s，3H)。 Combine 5-methylpyrazine-2-carboxylic acid (2.00g, 14.5mmol), 1-hydroxybenzotriazole (391mg, 2.90mmol), 1-(3-dimethylaminopropyl)-3-ethyl Carbodiimide hydrochloride (1.69g, 17.4mmol) was dissolved in anhydrous dichloromethane (10mL) and chloroform (30mL), under nitrogen protection, slowly added triethylamine (1.76g, 17.4mmol) at 0℃ ), the reaction solution was stirred at 25°C for 12 hours. The reaction was quenched by adding water (50 mL). The reaction solution was extracted with ethyl acetate (50mL x 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (20:1 petroleum ether/ethyl acetate, Rf =0.1) N -methoxy- N ,5-dimethylpyrazine-2-carboxamide (2.00 g, yellow oil) was obtained, and the yield was 76%. 1 H NMR: (400 MHz, CDCl 3 ) δ 8.80 (s, 1H), 8.45 (s, 1H), 3.73 (s, 3H), 3.40 (s, 3H), 2.61 (s, 3H).

第二步。 The second step.

1-(5-甲基吡嗪-2-基)乙酮。 1-(5-Methylpyrazin-2-yl)ethanone.

將N-甲氧基-N，5-二甲基吡嗪-2-甲醯胺(1.50g，8.28mmol)溶於四氫呋喃(30mL)，0℃下滴加甲基溴化鎂(3M乙醚溶液，13.3mL，39.9mmol)，後於25℃攪拌1小時。混合物冷却到0℃，加入水(10mL)淬滅反應。混合物用乙酸乙酯(30mL x 3)萃取，無水硫酸鈉乾燥後減壓濃縮。剩餘物用矽膠柱色譜法純化(20：1石油醚/乙酸乙酯，Rf=0.2)得到1-(5-甲基吡嗪-2-基)乙(700mg，黃色油狀物)，產率：62%。MS-ESI計算值[M+H]+ 137，實測值137。 Dissolve N -methoxy- N ,5-dimethylpyrazine-2-carboxamide (1.50g, 8.28mmol) in tetrahydrofuran (30mL), add methylmagnesium bromide (3M ether solution) dropwise at 0°C , 13.3mL, 39.9mmol), and then stirred at 25°C for 1 hour. The mixture was cooled to 0°C, and water (10 mL) was added to quench the reaction. The mixture was extracted with ethyl acetate (30 mL x 3), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (20:1 petroleum ether/ethyl acetate, Rf=0.2) to obtain 1-(5-methylpyrazin-2-yl)ethyl (700mg, yellow oil), yield : 62%. MS-ESI calculated value [M+H] + 137, measured value 137.

第三步。 third step.

1-(5(溴甲基)吡嗪-2-基)乙酮。 1-(5(Bromomethyl)pyrazin-2-yl)ethanone.

將1-(5-甲基吡嗪-2-基)乙(700mg，5.14mmol)溶於四氯化碳(20mL)中，然後加入偶氮二異丁腈(169mg，1.03mmol)和N-溴代丁 二醯亞胺(1.14g，6.43mmol)。反應液在100℃氮氣保護下反應5小時。反應液直接濾液並減壓濃縮，剩餘物用矽膠柱色譜法純化(20：1石油醚/乙酸乙酯，Rf=0.5)得到1-(5(溴甲基)吡嗪-2-基)乙(300mg，黃色油狀物)，產率：27%。MS-ESI計算值[M+H]+ 215和217，實測值215和217。 Dissolve 1-(5-methylpyrazin-2-yl) ethyl (700mg, 5.14mmol) in carbon tetrachloride (20mL), then add azobisisobutyronitrile (169mg, 1.03mmol) and N- Bromosuccinimide (1.14 g, 6.43 mmol). The reaction solution was reacted at 100°C under nitrogen protection for 5 hours. The reaction solution was directly filtrated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (20:1 petroleum ether/ethyl acetate, Rf=0.5) to obtain 1-(5(bromomethyl)pyrazin-2-yl)ethyl (300mg, yellow oil), yield: 27%. MS-ESI calculated values [M+H] + 215 and 217, and measured values 215 and 217.

第四步。 the fourth step.

1-((5-乙醯基吡嗪-2-基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮 1-((5-Acetylpyrazin-2-yl)methyl)-3,7-dimethyl-1 H -purine-2,6(3 H ,7 H )-dione

將1-(5(溴甲基)吡嗪-2-基)乙(300mg，1.40mmol)，3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(251mg，1.40mmol)，碘化鉀(23.2mg，0.140mmol)和碳酸鉀(578mg，4.19mmol)溶於無水N，N-二甲基甲醯胺(20mL)中。反應液加熱至120℃，反應3小時。反應液冷却至20℃，過濾，濾液減壓濃縮，剩餘物用矽膠柱色譜法純化(乙酸乙酯，Rf=0.3)得到1-((5-乙醯基吡嗪-2-基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(300mg，黃色固體)，產率：68%。MS ESI計算值[M+H]+ 315，實測值315。 1- (5 (bromomethyl) pyrazin-2-yl) acetate (300mg, 1.40mmol), 3,7- dimethyl -1 H - purine -2,6 (3 H, 7 H) - two Ketone (251 mg, 1.40 mmol), potassium iodide (23.2 mg, 0.140 mmol) and potassium carbonate (578 mg, 4.19 mmol) were dissolved in anhydrous N , N -dimethylformamide (20 mL). The reaction solution was heated to 120°C and reacted for 3 hours. The reaction solution was cooled to 20°C, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate, Rf=0.3) to obtain 1-((5-acetylpyrazin-2-yl)methyl ) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (300 mg of, yellow solid), yield: 68%. MS ESI calculated value [M+H] + 315, measured value 315.

第五步。 the fifth step.

3，7-二甲基-1-((5(1，1，1-三氟-2-羥基丙烷-2-基)吡嗪-2-基)甲基)-嘌呤-2，6(3H，7H)-二酮。 3,7-Dimethyl-1-((5(1,1,1-trifluoro-2-hydroxypropan-2-yl)pyrazin-2-yl)methyl)-purine-2,6(3 H , 7 H )-diketone.

將1-((5-乙醯基吡嗪-2-基)甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(300mg，0.954mmol)，氟化銫(14.5mg，0.0954mmol)溶於無水四氫呋喃(10mL)中，然後加入三甲基矽三氟甲基(407mg，2.86mmol)。反應液在25℃氮氣保護下反應2小時。然後加入鹽酸(4N，4mL)。混合物在室溫氮氣保護下反應1小時。加入碳酸氫鈉飽和溶液(10mL)淬 滅反應，用乙酸乙酯萃取(10 x 3mL)，有機相用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，剩餘物用矽膠柱色譜法純化(1：1石油醚/乙酸乙酯，Rf=0.3)得到3，7-二甲基-1-((5(1，1，1-三氟-2-羥基丙烷-2-基)吡嗪-2-基)甲基)-嘌呤-2，6(3H，7H)-二酮(100mg，白色固體)，產率：40%。1H NMR：(400MHz，Methonal-d 4 )δ8.85(s，1H)，8.65(s，1H)，7.92(s，1H)，5.40(s，2H)，3.99(s，3H)，3.56(s，3H)，1.78(s，3H)。MS ESI計算值[M+H]+ 385，實測值385。 1 - ((5-acetyl-2-yl) methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (300mg, 0.954mmol), cesium fluoride (14.5mg, 0.0954mmol) was dissolved in anhydrous tetrahydrofuran (10mL), and then trimethylsilyltrifluoromethyl (407mg, 2.86mmol) was added. The reaction solution was reacted for 2 hours at 25°C under nitrogen protection. Then hydrochloric acid (4N, 4mL) was added. The mixture was reacted for 1 hour under the protection of nitrogen at room temperature. The reaction was quenched by adding saturated sodium bicarbonate solution (10 mL), extracted with ethyl acetate (10 x 3 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (1: 1 petroleum ether/ethyl acetate, Rf=0.3) to obtain 3,7-dimethyl-1-((5(1,1,1-trifluoro-2-hydroxypropan-2-yl)pyrazine-2- yl) methyl) - purin -2,6 (3 H, 7 H) - dione (100 mg, white solid), yield: 40%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 8.85 (s, 1H), 8.65 (s, 1H), 7.92 (s, 1H), 5.40 (s, 2H), 3.99 (s, 3H), 3.56 (s, 3H), 1.78 (s, 3H). MS ESI calculated value [M+H] + 385, measured value 385.

實施例31。 Example 31.

1-((3-(1，1，1，3，3，3-六氟-2-羥基丙烷-2-基)異噁唑-5-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((3-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)isoxazol-5-yl)methyl)-3,7-dimethyl -1 H -purine-2,6-(3 H ,7 H )-dione.

Figure 105135042-A0305-02-0105-99
Figure 105135042-A0305-02-0105-99

第一步。 first step.

甲基5-(溴甲基)異噁唑-3-羧酸乙酯。 Methyl 5-(bromomethyl)isoxazole-3-carboxylic acid ethyl ester.

將甲基5-甲基異噁唑-3-羧酸乙酯(5.00g，35.4mmol)，N-溴代丁二醯亞胺(6.31g，35.4mmol)，過氧化苯甲醯(858mg，3.54mmol)溶於四氯化碳(20mL)中，80℃反應12小時。加入飽和硫代硫酸鈉溶液(30mL)淬滅反應。用二氯甲烷萃取(20mL x 3)，無水硫酸鈉乾燥，過濾， 濾液減壓濃縮，用矽膠柱色譜法分離純化(3：1石油醚/乙酸乙酯，Rf值=0.5)得到甲基5-(溴甲基)異噁唑-3-羧酸乙酯(2.00g，黃色油狀)，產率：26%。1H NMR：(400MHz，Methonal-d 4 )δ6.88(s，1H)，4.73(s，2H)，3.97(s，3H)。MS-ESI計算值[M+H]+ 220，222，實測值220，222。 Ethyl 5-methylisoxazole-3-carboxylate (5.00g, 35.4mmol), N -bromosuccinimide (6.31g, 35.4mmol), benzyl peroxide (858mg, 3.54mmol) was dissolved in carbon tetrachloride (20mL) and reacted at 80°C for 12 hours. The reaction was quenched by adding saturated sodium thiosulfate solution (30 mL). Extract with dichloromethane (20mL x 3), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and separate and purify by silica gel column chromatography (3:1 petroleum ether/ethyl acetate, Rf value=0.5) to obtain methyl 5 -(Bromomethyl)isoxazole-3-carboxylic acid ethyl ester (2.00 g, yellow oil), yield: 26%. 1 H NMR: (400 MHz, Methonal- d 4 ) δ 6.88 (s, 1H), 4.73 (s, 2H), 3.97 (s, 3H). MS-ESI calculated value [M+H] + 220,222, measured value 220,222.

第二步。 The second step.

甲基5-((3，7-二甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)異噁唑-3-羧酸乙酯。 Methyl 5-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)methyl)isoxazole-3 -Ethyl carboxylate.

將5-(溴甲基)異噁唑-3-羧酸乙酯(2.00g，9.09mmol)，3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(1.64g，9.09mmol)，碘化鉀(151mg，0.909mmol)和碳酸鉀(2.51g，18.2mmol)溶於N，N-二甲基甲醯胺(50mL)中。反應液升溫至120℃，攪拌3小時。冷却至室溫，過濾，濾液減壓濃縮用矽膠柱色譜法分離純化(乙酸乙酯，Rf值=0.4)得到甲基5-((3，7-二甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)異噁唑-3-羧酸乙酯(1.70g，黃色固體)，產率：59%。1H NMR：(400MHz，Methonal-d 4 )δ8.06(s，1H)，6.82(s，1H)，5.22(s，2H)，3.87(s，3H)，3.83(s，3H)，3.45(s，3H)。MS-ESI計算值[M+H]+ 320，實測值320。 5- (bromomethyl) isoxazole-3-carboxylate (2.00g, 9.09mmol), 3,7- dimethyl -1 H - purine -2,6- (3 H, 7 H) -Dione (1.64 g, 9.09 mmol), potassium iodide (151 mg, 0.909 mmol) and potassium carbonate (2.51 g, 18.2 mmol) were dissolved in N , N -dimethylformamide (50 mL). The reaction solution was heated to 120°C and stirred for 3 hours. Cooled to room temperature, filtered, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (ethyl acetate, Rf value = 0.4) to obtain methyl 5-((3,7-dimethyl-2,6-dioxo) -2,3,6,7-Tetrahydro- 1H -purin-1-yl)methyl)isoxazole-3-carboxylic acid ethyl ester (1.70 g, yellow solid), yield: 59%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 8.06 (s, 1H), 6.82 (s, 1H), 5.22 (s, 2H), 3.87 (s, 3H), 3.83 (s, 3H), 3.45 (s, 3H). MS-ESI calculated value [M+H] + 320, measured value 320.

第三步。 third step.

1-((3-(1，1，1，3，3，3-六氟-2-羥基丙烷-2-基)異噁唑-5-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮 1-((3-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)isoxazol-5-yl)methyl)-3,7-dimethyl -1 H -purine-2,6-(3 H ,7 H )-dione

將甲基5-((3，7-二甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)甲基)異噁唑-3-羧酸乙酯(200mg，0.626mmol)，氟化銫(95.0mg，0.626mmol)溶於四氫呋喃(10mL)中，室溫下加入三甲基三氟甲基矽烷(445mg，3.13mmol)，攪拌12小時。加入1N鹽酸(10mL)室溫下攪拌1小時，加入飽和碳酸氫鈉(50mL)淬滅反應。用乙酸乙酯萃取(10mL x 3)。合併有機相，用飽和食鹽水(30mL)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用製備型高效液相色譜純化，得到1-((3-(1，1，1，3，3，3-六氟-2-羥基丙烷-2-基)異噁唑-5-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(10.0mg，黃色固體)，產率：4%。1H NMR：(400MHz，Methonal-d 4 )δ7.95(s，1H)，6.52(s，1H)，5.37(s，2H)，4.00(s，3H)，3.57(s，3H)。MS-ESI計算值[M+H]+ 428，實測值428。 The methyl 5-((3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)methyl)isoxazole- Ethyl 3-carboxylate (200mg, 0.626mmol), cesium fluoride (95.0mg, 0.626mmol) were dissolved in tetrahydrofuran (10mL), trimethyltrifluoromethylsilane (445mg, 3.13mmol) was added at room temperature, Stir for 12 hours. 1N hydrochloric acid (10 mL) was added and stirred at room temperature for 1 hour, and saturated sodium bicarbonate (50 mL) was added to quench the reaction. Extract with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purified by preparative high performance liquid chromatography to obtain 1-((3-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)isoxazol-5-yl)methane yl) -3,7-dimethyl -1 H - purine -2,6- (3 H, 7 H) - dione (10.0 mg, yellow solid), yield: 4%. 1 H NMR: (400 MHz, Methonal- d 4 ) δ 7.95 (s, 1H), 6.52 (s, 1H), 5.37 (s, 2H), 4.00 (s, 3H), 3.57 (s, 3H). MS-ESI calculated value [M+H] + 428, measured value 428.

實施例32。 Example 32.

3，7-二甲基-1-((3-(1，1，1-三氟-2-羥基丙烷-2-基)異噁唑-5-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3,7-Dimethyl-1-((3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)isoxazol-5-yl)methyl)-1 H -purine- 2,6-(3 H ,7 H )-dione.

Figure 105135042-A0305-02-0107-100
Figure 105135042-A0305-02-0107-100

第一步。 first step.

1-(5-甲基異噁唑-3-基)乙酮。 1-(5-Methylisoxazol-3-yl)ethanone.

將甲基5-甲基異噁唑-3-羧酸乙酯(5.00g，35.4mmol)和三乙胺(21.5g，213mmol)溶於四氫呋喃(80mL)中，0℃下加入甲基溴化鎂(3M乙醚溶液，35mL，105mmol)，反應3小時。加入飽和氯化銨(30mL)淬滅反應。用乙酸乙酯萃取(30mL x 3)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法分離純化(3：1石油醚/乙酸乙酯，Rf值=0.7) 得到1-(5-甲基異噁唑-3-基)乙酮(1.00g，黃色油狀)，產率：23%。1H NMR：(400MHz，Methonal-d 4 )δ6.39(s，1H)，2.58(s，3H)，2.49(s，3H)。MS-ESI計算值[M+H]+ 126，實測值126。 Dissolve methyl 5-methylisoxazole-3-carboxylic acid ethyl ester (5.00 g, 35.4 mmol) and triethylamine (21.5 g, 213 mmol) in tetrahydrofuran (80 mL), and add methyl bromide at 0°C Magnesium (3M ether solution, 35 mL, 105 mmol), react for 3 hours. The reaction was quenched by adding saturated ammonium chloride (30 mL). Extract with ethyl acetate (30mL x 3), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and separate and purify by silica gel column chromatography (3:1 petroleum ether/ethyl acetate, Rf value=0.7) to obtain 1-( 5-Methylisoxazol-3-yl)ethanone (1.00 g, yellow oil), yield: 23%. 1 H NMR: (400 MHz, Methonal- d 4 ) δ 6.39 (s, 1H), 2.58 (s, 3H), 2.49 (s, 3H). MS-ESI calculated value [M+H] + 126, measured value 126.

第二步。 The second step.

1-(5-(溴甲基)異噁唑-3-基)乙酮。 1-(5-(Bromomethyl)isoxazol-3-yl)ethanone.

將1-(5-甲基異噁唑-3-基)乙酮(100mg，0.799mmol)，N-溴代丁二醯亞胺(142mg，0.799mmol)，過氧化苯甲醯(19.3mg，0.0800mmol)溶於四氯化碳(10mL)中，90℃反應12小時。加入飽和硫代硫酸鈉溶液(30mL)淬滅反應。用二氯甲烷萃取(10mL x 3)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到1-(5-(溴甲基)異噁唑-3-基)乙酮(150mg，黃色油狀)，產率：93%。MS-ESI計算值[M+H]+ 204和206，實測值204和206。 1-(5-Methylisoxazol-3-yl)ethanone (100mg, 0.799mmol), N -bromosuccinimide (142mg, 0.799mmol), benzyl peroxide (19.3mg, 0.0800mmol) was dissolved in carbon tetrachloride (10mL) and reacted at 90°C for 12 hours. The reaction was quenched by adding saturated sodium thiosulfate solution (30 mL). Extract with dichloromethane (10mL x 3), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain 1-(5-(bromomethyl)isoxazol-3-yl)ethanone (150mg, yellow oil) , Yield: 93%. MS-ESI calculated values [M+H] + 204 and 206, and measured values 204 and 206.

第三步。 third step.

1-((3-乙醯基異噁唑-5-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((3-Acetylisoxazol-5-yl)methyl)-3,7-dimethyl- 1H -purine-2,6-( 3H , 7H )-dione.

將1-(5-(溴甲基)異噁唑-3-基)乙酮(150mg，0.735mmol)，3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(132mg，0.735mmol)，碘化鉀(61.0mg，0.367mmol)和碳酸鉀(305mg，2.21mmol)溶於N，N-二甲基甲醯胺(10mL)中。反應液升溫至120℃，攪拌3小時。冷却至室溫，過濾，濾液減壓濃縮，用製備TLC板分離純化(乙酸乙酯，Rf值=0.3)，得到1-((3-乙醯基異噁唑-5-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(50.0mg，黃色固體)，產率：22%。MS-ESI計算值[M+H]+ 304，實測值304。 The 1-(5-(bromomethyl)isoxazol-3-yl)ethanone (150mg, 0.735mmol), 3,7-dimethyl- 1H -purine-2,6-( 3H ,7 H )-diketone (132 mg, 0.735 mmol), potassium iodide (61.0 mg, 0.367 mmol) and potassium carbonate (305 mg, 2.21 mmol) were dissolved in N , N -dimethylformamide (10 mL). The reaction solution was heated to 120°C and stirred for 3 hours. Cooled to room temperature, filtered, the filtrate was concentrated under reduced pressure, and separated and purified with a preparative TLC plate (ethyl acetate, Rf value = 0.3) to obtain 1-((3-acetylisoxazol-5-yl)methyl) 3,7-dimethyl -1 H - purine -2,6- (3 H, 7 H) - dione (50.0 mg, yellow solid), yield: 22%. MS-ESI calculated value [M+H] + 304, measured value 304.

第四步。 the fourth step.

3，7-二甲基-1-((3-(1，1，1-三氟-2-羥基丙烷-2-基)異噁唑-5-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮 3,7-Dimethyl-1-((3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)isoxazol-5-yl)methyl)-1 H -purine- 2,6-(3 H ,7 H )-dione

將1-[(3-乙醯基異噁唑-5-基)甲基]-3，7-二甲基嘌呤-2，6-二酮(50.0mg，0.164mmol)，氟化銫(25.0mg，0.164mmol)溶於四氫呋喃(10mL)中，室溫下加入三甲基三氟甲基矽烷(70.3mg，0.494mmol)，攪拌12小時。加入1N鹽酸(10mL)室溫下攪拌1小時，加入飽和碳酸氫鈉(50mL)淬滅反應。用乙酸乙酯萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用製備型高效液相色譜純化，得到3，7-二甲基-1-((3-(1，1，1-三氟-2-羥基丙烷-2-基)異噁唑-5-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮(22.0mg，黃色固體)，產率：36%。1H NMR：(400MHz，Methonal-d 4 )δ7.98(s，1H)，6.48(s，1H)，5.33(s，2H)，4.01(s，3H)，3.57(s，3H)，1.71(s，3H)。MS-ESI計算值[M+H]+ 374，實測值374。 1-[(3-Acetylisoxazol-5-yl)methyl]-3,7-dimethylpurine-2,6-dione (50.0mg, 0.164mmol), cesium fluoride (25.0 mg, 0.164 mmol) was dissolved in tetrahydrofuran (10 mL), trimethyltrifluoromethylsilane (70.3 mg, 0.494 mmol) was added at room temperature, and the mixture was stirred for 12 hours. 1N hydrochloric acid (10 mL) was added and stirred at room temperature for 1 hour, and saturated sodium bicarbonate (50 mL) was added to quench the reaction. Extract with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purified by preparative high performance liquid chromatography to obtain 3,7-dimethyl-1-((3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)isoxazol-5-yl ) methyl) -1 H - purine -2,6- (3 H, 7 H) - dione (22.0 mg, yellow solid), yield: 36%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.98 (s, 1H), 6.48 (s, 1H), 5.33 (s, 2H), 4.01 (s, 3H), 3.57 (s, 3H), 1.71 (s, 3H). MS-ESI calculated value [M+H] + 374, measured value 374.

實施例33。 Example 33.

3，7-二甲基-1-((2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-4-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3,7-Dimethyl-1-((2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl)methyl)-1 H -purine-2, 6-(3 H , 7 H )-dione.

Figure 105135042-A0305-02-0109-101
Figure 105135042-A0305-02-0109-101

第一步。 first step.

1-(4-(溴甲基)噻唑-2-基)乙酮。 1-(4-(Bromomethyl)thiazol-2-yl)ethanone.

將1-(4-甲基噻唑-2-基)乙酮(200mg，1.42mmol)，N-溴代丁二醯亞胺(252mg，1.42mmol)，偶氮二異丁腈(46.6mg，0.284mmol)溶於四氯化碳(20mL)中，80℃反應12小時。加入飽和硫代硫酸鈉溶液(30mL)淬滅反應。用二氯甲烷萃取(10mL x 3)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到1-(4-(溴甲基)噻唑-2-基)乙酮(200mg，黃色油狀)，產率：64%。1H NMR：(400MHz，Methonal-d 4 )δ7.97(s，1H)，4.73(s，2H)，2.66(s，3H)。MS-ESI計算值[M+H]+ 220，222，實測值220，222。 The 1-(4-methylthiazol-2-yl)ethanone (200mg, 1.42mmol), N -bromosuccinimide (252mg, 1.42mmol), azobisisobutyronitrile (46.6mg, 0.284 mmol) was dissolved in carbon tetrachloride (20 mL) and reacted at 80°C for 12 hours. The reaction was quenched by adding saturated sodium thiosulfate solution (30 mL). Extract with dichloromethane (10mL x 3), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain 1-(4-(bromomethyl)thiazol-2-yl)ethanone (200mg, yellow oil). Yield: 64%. 1 H NMR: (400 MHz, Methonal- d 4 ) δ 7.97 (s, 1H), 4.73 (s, 2H), 2.66 (s, 3H). MS-ESI calculated value [M+H] + 220,222, measured value 220,222.

第二步。 The second step.

1-((2-乙醯基噻唑-4-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((2-Acetylthiazol-4-yl)methyl)-3,7-dimethyl- 1H -purine-2,6-( 3H , 7H )-dione.

將1-(4-(溴甲基)噻唑-2-基)乙酮(100mg，0.454mmol)，3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(81.9mg，0.454mmol)，碘化鉀(7.50mg，0.0454mmol)和碳酸鉀(125mg，0.908mmol)溶於N，N-二甲基甲醯胺(10mL)中。反應液升溫至120℃，攪拌3小時。冷却至室溫，過濾，濾液減壓濃縮用製備TLC板分離純化(乙酸乙酯，Rf值=0.3)得到1-((2-乙醯基噻唑-4-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(80.0mg，黃色固體)，產率：55%。1H NMR：(400MHz，Methonal-d 4 )δ7.92(s，1H)，7.73(s，1H)，5.38(s，2H)，4.00(s，3H)，3.57(s，3H)，2.64(s，3H)。MS-ESI計算值[M+H]+ 320，實測值320。 1- (4- (bromomethyl) thiazol-2-yl) ethanone (100mg, 0.454mmol), 3,7- dimethyl -1 H - purine -2,6- (3 H, 7 H) -Dione (81.9 mg, 0.454 mmol), potassium iodide (7.50 mg, 0.0454 mmol) and potassium carbonate (125 mg, 0.908 mmol) were dissolved in N , N -dimethylformamide (10 mL). The reaction solution was heated to 120°C and stirred for 3 hours. Cool to room temperature, filter, and concentrate the filtrate under reduced pressure. Separate and purify with a preparative TLC plate (ethyl acetate, Rf value=0.3) to obtain 1-((2-acetylthiazol-4-yl)methyl)-3,7 - dimethyl -1 H - purine -2,6- (3 H, 7 H) - dione (80.0 mg, yellow solid), yield: 55%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.92 (s, 1H), 7.73 (s, 1H), 5.38 (s, 2H), 4.00 (s, 3H), 3.57 (s, 3H), 2.64 (s, 3H). MS-ESI calculated value [M+H] + 320, measured value 320.

第三步。 third step.

3，7-二甲基-1-((2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-4-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3,7-Dimethyl-1-((2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl)methyl)-1 H -purine-2, 6-(3 H , 7 H )-dione.

將1-((2-乙醯基噻唑-4-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(200mg，0.626mmol)，氟化銫(95.0mg，0.626mmol)溶於四氫呋喃(10mL)中，室溫下加入三甲基-三氟甲基-矽烷(267mg，1.88mmol)，攪拌12小時。加入水(20mL)淬滅反應。用乙酸乙酯萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用製備型高效液相色譜純化，得到3，7-二甲基-1-((2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-4-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮(100mg，黃色固體)，產率：41%。1H NMR：(400MHz，Methonal-d 4 )δ8.10(s，1H)，7.33(s，1H)，5.32(s，2H)，4.03(s，3H)，3.57(s，3H)，1.80(s，3H)。MS-ESI計算值[M+H]+ 390，實測值390。 The 1-((2-acetylthiazol-4-yl)methyl)-3,7-dimethyl- 1H -purine-2,6-( 3H , 7H )-dione (200mg, 0.626mmol), cesium fluoride (95.0mg, 0.626mmol) was dissolved in tetrahydrofuran (10mL), trimethyl-trifluoromethyl-silane (267mg, 1.88mmol) was added at room temperature, and stirred for 12 hours. The reaction was quenched by adding water (20 mL). Extract with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purified by preparative high performance liquid chromatography to obtain 3,7-dimethyl-1-((2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl)methan Base)-1 H -purine-2,6-( 3H , 7H )-dione (100mg, yellow solid), yield: 41%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 8.10 (s, 1H), 7.33 (s, 1H), 5.32 (s, 2H), 4.03 (s, 3H), 3.57 (s, 3H), 1.80 (s, 3H). MS-ESI calculated value [M+H] + 390, measured value 390.

實施例34。 Example 34.

3，7-二甲基-1-((5-甲基-2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-4-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3,7-Dimethyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl)methyl)-1 H -Purine-2,6-( 3H , 7H )-dione.

Figure 105135042-A0305-02-0111-103
Figure 105135042-A0305-02-0111-103

第一步。 first step.

1-(4-(溴甲基)-5-甲基噻唑-2-基)乙酮。 1-(4-(Bromomethyl)-5-methylthiazol-2-yl)ethanone.

將1-(4，5-二甲基吡啶-2-基)乙酮(200mg，1.29mmol)，N-溴代丁二醯亞胺(229mg，1.29mmol)，偶氮二異丁腈(21.1mg，0.129mmol)溶於四氯化碳(10mL)中，80℃反應12小時。加入飽和硫代硫酸鈉溶液(30mL)淬滅反應。用二氯甲烷萃取(10mL x 3)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到1-(4-(溴甲基)-5-甲基噻唑-2-基)乙酮(200mg，黃色油狀)，產率：66%。1H NMR：(400MHz，Methonal-d 4 )δ4.88(s，2H)，2.65(s，3H)，2.47(s，3H)。MS-ESI計算值[M+H]+ 234，236，實測值234，236。 The 1-(4,5-dimethylpyridin-2-yl)ethanone (200mg, 1.29mmol), N -bromosuccinimide (229mg, 1.29mmol), azobisisobutyronitrile (21.1 mg, 0.129mmol) was dissolved in carbon tetrachloride (10mL) and reacted at 80°C for 12 hours. The reaction was quenched by adding saturated sodium thiosulfate solution (30 mL). It was extracted with dichloromethane (10mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 1-(4-(bromomethyl)-5-methylthiazol-2-yl)ethanone (200mg, Yellow oil), yield: 66%. 1 H NMR: (400 MHz, Methonal- d 4 ) δ 4.88 (s, 2H), 2.65 (s, 3H), 2.47 (s, 3H). MS-ESI calculated value [M+H] + 234,236, measured value 234,236.

第二步。 The second step.

1-((2-乙醯基-5-甲基噻唑-4-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((2-Acetyl-5-methylthiazol-4-yl)methyl)-3,7-dimethyl-1 H -purine-2,6-(3 H ,7 H )-di ketone.

將1-(4-(溴甲基)-5-甲基噻唑-2-基)乙酮(200mg，0.854mmol)，3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(154mg，0.854mmol)，碘化鉀(14.0mg，0.0854mmol)和碳酸鉀(354mg，2.56mmol)溶於N，N-二甲基甲醯胺(10mL)中。反應液升溫至120℃，攪拌3小時。冷却至室溫，過濾，濾液減壓濃縮用製備TLC板分離純化(乙酸乙酯，Rf值=0.3)得到1-((2-乙醯基-5-甲基噻唑-4-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(200mg，黃色固體)，產率：70%。1H NMR：(400MHz，Methonal-d 4 )δ7.90(s，1H)，5.35(s，2H)，4.00(s，3H)，3.55(s，3H)，2.66(s，3H)，2.61(s，3H)。MS-ESI計算值[M+H]+ 334，實測值334。 The 1-(4-(bromomethyl)-5-methylthiazol-2-yl)ethanone (200mg, 0.854mmol), 3,7-dimethyl- 1H -purine-2,6-(3 H, 7 H) - dione (154mg, 0.854mmol), potassium iodide (14.0mg, 0.0854mmol) and potassium carbonate (354mg, 2.56mmol) was dissolved in N, N - dimethylformamide (10 mL) in. The reaction solution was heated to 120°C and stirred for 3 hours. Cool to room temperature, filter, and concentrate the filtrate under reduced pressure. Separate and purify with a preparative TLC plate (ethyl acetate, Rf value=0.3) to obtain 1-((2-acetyl-5-methylthiazol-4-yl)methyl )-3,7-Dimethyl- 1H -purine-2,6-( 3H , 7H )-dione (200mg, yellow solid), yield: 70%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.90 (s, 1H), 5.35 (s, 2H), 4.00 (s, 3H), 3.55 (s, 3H), 2.66 (s, 3H), 2.61 (s, 3H). MS-ESI calculated value [M+H] + 334, measured value 334.

第三步。 third step.

3，7-二甲基-1-((5-甲基-2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-4-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3,7-Dimethyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl)methyl)-1 H -Purine-2,6-( 3H , 7H )-dione.

將1-((2-乙醯基-5-甲基噻唑-4-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(80.0mg，0.240mmol)，氟化銫(18.2mg，0.120mmol)溶於四氫呋喃(10mL)中，室溫下加入三甲基-三氟甲基-矽烷(102mg，0.720mmol)，攪拌12小時。加入水(20mL)淬滅反應。用乙酸乙酯萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用製備型高效液相色譜純化，得到3，7-二甲基-1-((5-甲基-2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-4-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮(35.0mg，黃色固體)，產率：36%。1H NMR：(400MHz，Methonal-d 4 )δ8.27(s，1H)，5.36(s，2H)，4.06(s，3H)，3.57(s，3H)，2.73(s，3H)，1.90(s，3H)。MS-ESI計算值[M+H]+ 404，實測值404。 1-((2-Acetyl-5-methylthiazol-4-yl)methyl)-3,7-dimethyl- 1H -purine-2,6-( 3H , 7H )- Dione (80.0mg, 0.240mmol), cesium fluoride (18.2mg, 0.120mmol) were dissolved in tetrahydrofuran (10mL), added trimethyl-trifluoromethyl-silane (102mg, 0.720mmol) at room temperature, and stirred 12 hours. The reaction was quenched by adding water (20 mL). Extract with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purified by preparative high performance liquid chromatography to obtain 3,7-dimethyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazole- 4- yl) methyl) -1 H - purine -2,6- (3 H, 7 H) - dione (35.0 mg, yellow solid), yield: 36%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 8.27 (s, 1H), 5.36 (s, 2H), 4.06 (s, 3H), 3.57 (s, 3H), 2.73 (s, 3H), 1.90 (s, 3H). MS-ESI calculated value [M+H] + 404, measured value 404.

實施例35。 Example 35.

3，7-二甲基-1-((2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-5-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3,7-Dimethyl-1-((2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-5-yl)methyl)-1 H -purine-2, 6-(3 H , 7 H )-dione.

Figure 105135042-A0305-02-0113-104
Figure 105135042-A0305-02-0113-104

第一步。 first step.

1-(5-甲基噻唑-2-基)乙基環己酮。 1-(5-Methylthiazol-2-yl)ethylcyclohexanone.

將5-甲基噻唑(2.00g，20.2mmol)溶於四氫呋喃(50mL)中，-78℃時，在氮氣保護下緩慢滴加正丁基鋰(2.5M四氫呋喃溶液，9.68mL，24.2mmol)。反應液-78℃下攪拌0.5小時，並緩慢滴加溶於四氫呋喃中(1mL)的N-甲氧基-N-甲基乙醯胺(2.50g，24.2mmol)。反應液升溫至0℃下攪拌1.5小時。在0℃下向反應液中緩慢加入水(10mL)，並用乙酸乙酯(30mL x 3)萃取。合併有機相，用無水硫酸鈉乾燥，過濾並減壓蒸餾，所得到產品用高效製備板純化(1：1石油醚/乙酸乙酯，Rf=0.7)得到產物1-(5-甲基噻唑-2-基)乙基環己酮(1.45g，黃色固體)，產率：51%。1H NMR：(400MHz，Methonal-d 4)δ7.73(s，1H)，2.61(s，3H)，2.57(s，3H)。MS-ESI計算值[M+H]+ 142，實測值142。 5-Methylthiazole (2.00g, 20.2mmol) was dissolved in tetrahydrofuran (50mL). At -78°C, n-butyllithium (2.5M tetrahydrofuran solution, 9.68mL, 24.2mmol) was slowly added dropwise under nitrogen protection. The reaction solution was stirred at -78°C for 0.5 hours, and N -methoxy- N -methylacetamide (2.50 g, 24.2 mmol) dissolved in tetrahydrofuran (1 mL) was slowly added dropwise. The reaction solution was heated to 0°C and stirred for 1.5 hours. Water (10 mL) was slowly added to the reaction solution at 0°C, and extracted with ethyl acetate (30 mL x 3). The organic phases were combined, dried with anhydrous sodium sulfate, filtered and distilled under reduced pressure. The product obtained was purified with a high-efficiency preparation plate (1:1 petroleum ether/ethyl acetate, Rf=0.7) to obtain the product 1-(5-methylthiazole 2-yl)ethylcyclohexanone (1.45 g, yellow solid), yield: 51%. 1 H NMR: (400 MHz, Methonal- d 4 ) δ 7.73 (s, 1H), 2.61 (s, 3H), 2.57 (s, 3H). MS-ESI calculated value [M+H] + 142, measured value 142.

第二步。 The second step.

1-(5-(溴甲基)噻唑-2-基)乙基環己酮。 1-(5-(Bromomethyl)thiazol-2-yl)ethylcyclohexanone.

將1-(5-甲基噻唑-2-基)乙基環己酮(200mg，1.42mmol)及偶氮異丁晴(2.33mg，0.0142mmol)溶於氯仿(5mL)中，室溫下加入溴代丁二醯亞胺(252mg，1.42mmol)。反應液加熱至78℃並攪拌16小時。反應液冷却至室溫，緩慢加入水(30mL)並用氯仿(30mL x 3)萃取。合併有機相，並用無水硫酸鈉乾燥，過濾，濾液減壓濃縮得到粗產品1-(5-(溴甲基)噻唑-2-基)乙基環己酮(290mg，黃色油狀)。MS-ESI計算值[M+H]+ 220和222，實測值220和222。 Dissolve 1-(5-methylthiazol-2-yl)ethylcyclohexanone (200mg, 1.42mmol) and azoisobutyronitrile (2.33mg, 0.0142mmol) in chloroform (5mL) and add at room temperature Bromosuccinimide (252 mg, 1.42 mmol). The reaction solution was heated to 78°C and stirred for 16 hours. The reaction solution was cooled to room temperature, water (30 mL) was slowly added and extracted with chloroform (30 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product 1-(5-(bromomethyl)thiazol-2-yl)ethylcyclohexanone (290 mg, yellow oil). MS-ESI calculated values [M+H] + 220 and 222, measured values 220 and 222.

第三步。 third step.

1-((2-乙醯基噻唑-5-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((2-Acetylthiazol-5-yl)methyl)-3,7-dimethyl- 1H -purine-2,6-( 3H , 7H )-dione.

將1-(5-(溴甲基)噻唑-2-基)乙基環己酮(290mg，1.05mmol)，3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(284mg，1.58mmol)及碘化鉀(17.5mg，0.105mmol)溶於N，N-二甲基甲醯胺(5mL)中，加入碳酸鉀(437mg，3.16mmol)，130℃反應2.5小時。反應液冷却至室溫，過濾，濾液減壓濃縮，得到的產品用高效製備板純化(乙酸乙酯，Rf=0.4)得到產物1-((2-乙醯基噻唑-5-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(292mg，黃色固體)，產率：87%。MS-ESI計算值[M+H]+ 320，實測值320。 The 1-(5-(bromomethyl)thiazol-2-yl)ethylcyclohexanone (290mg, 1.05mmol), 3,7-dimethyl-1H-purine-2,6-(3H, 7 H )-Diketone (284mg, 1.58mmol) and potassium iodide (17.5mg, 0.105mmol) were dissolved in N , N -dimethylformamide (5mL), potassium carbonate (437mg, 3.16mmol) was added, and the reaction was carried out at 130℃ for 2.5 hour. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The product obtained was purified with a high-efficiency preparation plate (ethyl acetate, Rf=0.4) to obtain the product 1-((2-acetylthiazol-5-yl)methyl ) -3,7-dimethyl -1 H - purine -2,6- (3 H, 7 H) - dione (292 mg, yellow solid), yield: 87%. MS-ESI calculated value [M+H] + 320, measured value 320.

第四步。 the fourth step.

3，7-二甲基-1-((2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-5-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3,7-Dimethyl-1-((2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-5-yl)methyl)-1 H -purine-2, 6-(3 H , 7 H )-dione.

將1-((2-乙醯基噻唑-5-基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(280mg，0.438mmol)及氟化銫(6.66mg，0.0438mmol)溶於四氫呋喃(6mL)中，在氮氣保護下緩慢加入三氟甲基三甲基矽烷(75.0mg，0.500mmol)。反應25℃下攪拌1.5小時。加入4N鹽酸水溶液(0.2mL)並室溫攪拌半小時後，用飽和碳酸氫鈉溶液(10mL)調節pH值至7，加入水(20mL)並用乙酸乙酯(50mL x 3)，有機相用無水硫酸鈉乾燥，減壓濃縮，得到粗產品用製備高效液相色譜純化得產物3，7-二甲基-1-((2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-5-基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮(32.0mg，白色固體)，產率：19%。1H NMR：(400MHz，Methonal-d 4)δ7.89(s，1H)，7.82(s，1H)，5.35(s，2H)，4.00(s，3H)，3.56(s，3H)，1.76(s，3H)。MS-ESI計算值[M+H]+ 390，實測值390。 1 - ((2-acetylamino-thiazol-5-yl) methyl) -3,7-dimethyl -1 H - purine -2,6- (3 H, 7 H) - dione (280mg, 0.438mmol) and cesium fluoride (6.66mg, 0.0438mmol) were dissolved in tetrahydrofuran (6mL), and trifluoromethyltrimethylsilane (75.0mg, 0.500mmol) was slowly added under the protection of nitrogen. The reaction was stirred at 25°C for 1.5 hours. After adding 4N aqueous hydrochloric acid (0.2mL) and stirring at room temperature for half an hour, adjust the pH to 7 with saturated sodium bicarbonate solution (10mL), add water (20mL) and use ethyl acetate (50mL x 3), and use anhydrous organic phase After drying over sodium sulfate and concentrating under reduced pressure, the crude product was obtained by preparative high performance liquid chromatography to obtain the product 3,7-dimethyl-1-((2-(1,1,1-trifluoro-2-hydroxypropane-2 - yl) thiazol-5-yl) methyl) -1 H - purine -2,6- (3 H, 7 H) - dione (32.0 mg [, white solid), yield: 19%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.89 (s, 1H), 7.82 (s, 1H), 5.35 (s, 2H), 4.00 (s, 3H), 3.56 (s, 3H), 1.76 (s, 3H). MS-ESI calculated value [M+H] + 390, measured value 390.

實施例36。 Example 36.

3，7-二甲基-1-(2-(4-甲基-2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-5-基)乙基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3,7-Dimethyl-1-(2-(4-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-5-yl)ethyl)- 1 H -purine-2,6-(3 H ,7 H )-dione.

Figure 105135042-A0305-02-0116-105
Figure 105135042-A0305-02-0116-105

第一步。 first step.

1-(5-(2-羥乙基)-4-甲基噻唑-2-基)乙酮。 1-(5-(2-hydroxyethyl)-4-methylthiazol-2-yl)ethanone.

將2-(4-甲基噻唑-5-基)乙醇(500mg，3.49mmol)溶於四氫呋喃(100mL)中，-78℃下加入正丁基鋰(3M正己烷溶液，2.33mL，6.98mmol)，反應半小時後加入N-甲氧基-N-甲基-乙醯胺(432mg，4.19mmol)，繼續攪拌3小時。加入飽和氯化銨(50mL)淬滅反應。乙酸乙酯萃取(10mL x 3)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法分離純化(5：1石油醚/乙酸乙酯，Rf值=0.1)，得到1-(5-(2-羥乙基)-4-甲基噻唑-2-基)乙酮(200mg，黃色油狀)，產率：31%。1H NMR：(400MHz，CDCl3)δ3.78(t，J=6.4Hz，2H)，3.18(t，J=6.4Hz，2H)，2.67(s，3H)，2.47(s，3H)。MS-ESI計算值[M+H]+ 186，實測值186。 Dissolve 2-(4-methylthiazol-5-yl)ethanol (500mg, 3.49mmol) in tetrahydrofuran (100mL), add n-butyllithium (3M n-hexane solution, 2.33mL, 6.98mmol) at -78°C After reacting for half an hour, N -methoxy- N -methyl-acetamide (432 mg, 4.19 mmol) was added, and stirring was continued for 3 hours. The reaction was quenched by adding saturated ammonium chloride (50 mL). Extract with ethyl acetate (10mL x 3), dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and separate and purify by silica gel column chromatography (5:1 petroleum ether/ethyl acetate, Rf value=0.1) to obtain 1-( 5-(2-hydroxyethyl)-4-methylthiazol-2-yl)ethanone (200 mg, yellow oil), yield: 31%. 1 H NMR: (400 MHz, CDCl 3 ) δ 3.78 (t, J = 6.4 Hz, 2H), 3.18 (t, J = 6.4 Hz, 2H), 2.67 (s, 3H), 2.47 (s, 3H). MS-ESI calculated value [M+H] + 186, measured value 186.

第二步。 The second step.

2-(2-乙醯基-4-甲基-噻唑-5-基)乙基甲磺酸酯。 2-(2-Acetyl-4-methyl-thiazol-5-yl) ethyl methanesulfonate.

將1-(5-(2-羥乙基)-4-甲基噻唑-2-基)乙酮(120mg，0.647mmol)和三乙胺(196mg，1.94mmol)溶於二氯甲烷(10mL)中，0℃下加入甲烷磺醯氯(148mg，1.30mmol)。反應液緩慢升至室溫，攪拌2小時。加入碳酸氫鈉水溶液(50mL)淬滅反應。用二氯甲烷萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用製備TLC板分離純化(1：1石油醚/乙酸乙酯，Rf=0.5)，得到2-(2-乙醯基-4-甲基-噻唑-5-基)乙基甲磺酸酯(150mg，黃色油狀)，產率：88%。1H NMR：(400MHz，CDCl3)δ4.41(t，J=6.4Hz，2H)，3.27(t，J=6.4Hz，2H)，3.01(s，3H)，2.67(s，3H)，2.46(s，3H)。MS-ESI計算值[M+H]+ 264，實測值264。 Dissolve 1-(5-(2-hydroxyethyl)-4-methylthiazol-2-yl)ethanone (120mg, 0.647mmol) and triethylamine (196mg, 1.94mmol) in dichloromethane (10mL) In, methanesulfonyl chloride (148mg, 1.30mmol) was added at 0°C. The reaction solution was slowly raised to room temperature and stirred for 2 hours. The reaction was quenched by the addition of aqueous sodium bicarbonate (50 mL). Extract with dichloromethane (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and separated and purified with a preparative TLC plate (1:1 petroleum ether/ethyl acetate, Rf=0.5) to obtain 2-(2- Acetyl-4-methyl-thiazol-5-yl) ethyl methanesulfonate (150 mg, yellow oil), yield: 88%. 1 H NMR: (400MHz, CDCl3) δ 4.41 (t, J = 6.4 Hz, 2H), 3.27 (t, J = 6.4 Hz, 2H), 3.01 (s, 3H), 2.67 (s, 3H), 2.46 (s, 3H). MS-ESI calculated value [M+H] + 264, measured value 264.

第三步。 third step.

1-(2-(2-乙醯基-4-甲基噻唑-5-基)乙基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-(2-(2-Acetyl-4-methylthiazol-5-yl)ethyl)-3,7-dimethyl-1 H -purine-2,6-(3 H ,7 H ) -Diketone.

將2-(2-乙醯基-4-甲基-噻唑-5-基)乙基甲磺酸酯(150mg，0.569mmol)，3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(102mg，0.569mmol)，碘化鉀(18.9mg，0.114mmol)和碳酸鉀(236mg，1.71mmol)溶於N，N-二甲基甲醯胺(10mL)中。反應液升溫至120℃，攪拌3小時。冷却至室溫，過濾，濾液減壓濃縮。用製備TLC板分離純化(乙酸乙酯，Rf值=0.5)，得到1-(2-(2-乙醯基-4-甲基噻唑-5-基)乙基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(30.0mg，黃色固體)，產率：15%。MS-ESI計算值[M+H]+ 348，實測值348。 The 2-(2-acetyl-4-methyl-thiazol-5-yl) ethyl methanesulfonate (150mg, 0.569mmol), 3,7-dimethyl- 1H -purine-2,6 - (3 H, 7 H) - dione (102mg, 0.569mmol), potassium iodide (18.9mg, 0.114mmol) and potassium carbonate (236mg, 1.71mmol) was dissolved in N, N - dimethylformamide (10 mL) in. The reaction solution was heated to 120°C and stirred for 3 hours. Cool to room temperature, filter, and concentrate the filtrate under reduced pressure. Separation and purification with a preparative TLC plate (ethyl acetate, Rf value = 0.5), to obtain 1-(2-(2-acetanyl-4-methylthiazol-5-yl)ethyl)-3,7-dimethyl yl -1 H - purine -2,6- (3 H, 7 H) - dione (30.0 mg, yellow solid), yield: 15%. MS-ESI calculated value [M+H] + 348, measured value 348.

第四步 the fourth step

3，7-二甲基-1-(2-(4-甲基-2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-5-基)乙基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3,7-Dimethyl-1-(2-(4-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-5-yl)ethyl)- 1 H -purine-2,6-(3 H ,7 H )-dione.

將1-(2-(2-乙醯基-4-甲基噻唑-5-基)乙基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(40.0mg，0.115mmol)，氟化銫(17.5mg，0.115mmol)溶於四氫呋喃(10mL)中，室溫下加入三甲基三氟甲基矽烷(49.0mg，0.345mmol)，攪拌12小時。加入水(20mL)淬滅反應。用乙酸乙酯萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用製備型高效液相色譜純化，得到3，7-二甲基-1-(2-(4-甲基-2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-5-基)乙基)-1H-嘌呤-2，6-(3H，7H)-二酮(15.0mg，黃色固體)，產率：31%。1H NMR：(400MHz，Methonal-d 4 )δ8.37(s，1H)，4.25(t，J=6.4Hz，2H)，4.01(s，3H)，3.54(s，3H)，3.26(t，J=6.4Hz，2H)，2.50(s，3H)，1.90(s，3H)。MS-ESI計算值[M+H]+ 418，實測值418。 1-(2-(2-Acetyl-4-methylthiazol-5-yl)ethyl)-3,7-dimethyl- 1H -purine-2,6-( 3H , 7H )-Diketone (40.0mg, 0.115mmol), cesium fluoride (17.5mg, 0.115mmol) dissolved in tetrahydrofuran (10mL), add trimethyltrifluoromethylsilane (49.0mg, 0.345mmol) at room temperature, Stir for 12 hours. The reaction was quenched by adding water (20 mL). Extract with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purified by preparative high performance liquid chromatography to obtain 3,7-dimethyl-1-(2-(4-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl) thiazol-5-yl) ethyl) -1 H - purine -2,6- (3 H, 7 H) - dione (15.0 mg, yellow solid), yield: 31%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 8.37 (s, 1H), 4.25 (t, J = 6.4 Hz, 2H), 4.01 (s, 3H), 3.54 (s, 3H), 3.26 (t , J =6.4Hz, 2H), 2.50(s, 3H), 1.90(s, 3H). MS-ESI calculated value [M+H] + 418, measured value 418.

實施例37。 Example 37.

1-(3-羥基-2-(羥甲基)-2-甲基丙基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-(3-Hydroxy-2-(hydroxymethyl)-2-methylpropyl)-3,7-dimethyl-1 H -purine-2,6-(3 H ,7 H )-dione .

Figure 105135042-A0305-02-0118-106
Figure 105135042-A0305-02-0118-106

將3，7-二甲基-1-[(3-甲基氧雜環丁烷-3-基)甲基]嘌呤-2，6-二酮(20.0mg，0.0757mmol)溶於0.16%鹽酸(0.5mL)，反應液於室溫下攪拌反應6小時，用飽和碳酸氫鈉水溶液調節pH至7，用高效液相色譜法純化，得1-(3-羥基-2-(羥甲基)-2-甲基丙基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(12.0mg，白色固體)，產率：56%。1H NMR：(400MHz， CDCl3)δ7.58(s，1H)，4.25-3.94(m，7H)，3.62(s，3H)，3.35-3.26(m，2H)，3.25-3.14(m，2H)，1.01(s，3H)。MS-ESI計算值[M+H]+ 283，實測值283。 Dissolve 3,7-dimethyl-1-[(3-methyloxetan-3-yl)methyl]purine-2,6-dione (20.0mg, 0.0757mmol) in 0.16% hydrochloric acid (0.5mL), the reaction solution was stirred at room temperature for 6 hours, adjusted to pH 7 with saturated aqueous sodium bicarbonate solution, and purified by high performance liquid chromatography to obtain 1-(3-hydroxy-2-(hydroxymethyl) 2-methylpropyl) -3,7-dimethyl -1 H - purine -2,6- (3 H, 7 H) - dione (12.0 mg, white solid), yield: 56%. 1 H NMR: (400MHz, CDCl 3 ) δ 7.58 (s, 1H), 4.25-3.94 (m, 7H), 3.62 (s, 3H), 3.35 to 3.26 (m, 2H), 3.25 to 3.14 (m, 2H), 1.01 (s, 3H). MS-ESI calculated value [M+H] + 283, measured value 283.

實施例38。 Example 38.

1-(2-(2-羥基-2-甲基環丙基)乙基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1- (2- (2-hydroxy-cyclopropyl) ethyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

Figure 105135042-A0305-02-0119-107
Figure 105135042-A0305-02-0119-107

第一步。 first step.

2-(2-(3，7-二甲基-2，6-二氧-2，3，6，7-四氫-1H-嘌呤-基)乙氧基)乙酸酯。 2-(2-(3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-yl)ethoxy)acetate.

室溫條件下，向氫化鈉(21.0mg，0.890mmol)的N，N-二甲基甲醯胺(10mL)溶液中加入1-(2-羥基乙基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(100mg，0.446mmol)，反應液在25℃攪拌1小時。再加入2-溴乙酸乙酯(149mg，0.890mmol)。反應液繼續攪拌16小時。過濾除去不溶物，濾液減壓濃縮，用製備高效液相色譜法分離純化得到2-(2-(3，7-二甲基-2，6-二氧-2，3，6，7-四氫-1H-嘌呤-基)乙氧基)乙酸酯(60.0mg，白色固體)，產率：43%。 At room temperature, to a solution of sodium hydride (21.0 mg, 0.890 mmol) in N , N -dimethylformamide (10 mL) was added 1-(2-hydroxyethyl)-3,7-dimethyl- 1 H - purine -2,6 (3 H, 7 H) - dione (100mg, 0.446mmol), the reaction was stirred at 25 ℃ 1 hour. Additional ethyl 2-bromoacetate (149 mg, 0.890 mmol) was added. The reaction solution was continuously stirred for 16 hours. The insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure, and separated and purified by preparative high performance liquid chromatography to obtain 2-(2-(3,7-dimethyl-2,6-diox-2,3,6,7-tetra Hydrogen-1 H -purin-yl)ethoxy)acetate (60.0 mg, white solid), yield: 43%.

MS-ESI計算值[M+H]+ 311，實測值311。 MS-ESI calculated value [M+H] + 311, measured value 311.

第二步。 The second step.

1-(2-(2-羥基-2-甲基環丙基)乙基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1- (2- (2-hydroxy-cyclopropyl) ethyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

在-78℃向2-(2-(3，7-二甲基-2，6-二氧-2，3，6，7-四氫-1H-嘌呤-基)乙氧基)乙酸酯(100mg，0.322mmol)的四氫呋喃(5mL)溶液緩慢滴加甲基溴化鎂溶液(3M四氫呋喃溶液，0.43mL，1.29mmol)。反應液在-78℃攪拌2小時。加入飽和氯化銨水溶液(20mL)淬滅反應。混合物用乙酸乙酯(20mL x 3)萃取。合併有機相，減壓濃縮，用製備高效液相色譜法分離純化，得到1-(2-(2-羥基-2-甲基環丙基)乙基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(40.0mg，無色油狀物)。產率：42%。1H NMR：(400MHz，Methonal-d 4 )δ7.88(s，1H)，4.23(t，J=5.8Hz，2H)，3.98(s，3H)，3.72(t，J=5.8Hz，2H)，3.53(s，3H)，3.32(s，2H)，1.13(s，6H)。MS-ESI計算值[M+H]+ 296，實測值296。 To 2-(2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -purin-yl)ethoxy)acetic acid at -78℃ A solution of the ester (100 mg, 0.322 mmol) in tetrahydrofuran (5 mL) was slowly added dropwise to a solution of methylmagnesium bromide (3M tetrahydrofuran solution, 0.43 mL, 1.29 mmol). The reaction solution was stirred at -78°C for 2 hours. The reaction was quenched by adding saturated aqueous ammonium chloride solution (20 mL). The mixture was extracted with ethyl acetate (20 mL x 3). The organic phases were combined, concentrated under reduced pressure, and separated and purified by preparative high performance liquid chromatography to obtain 1-(2-(2-hydroxy-2-methylcyclopropyl)ethyl)-3,7-dimethyl-1 H - purine -2,6 (3 H, 7 H) - dione (40.0mg, colorless oil). Yield: 42%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.88 (s, 1H), 4.23 (t, J = 5.8 Hz, 2H), 3.98 (s, 3H), 3.72 (t, J = 5.8 Hz, 2H ), 3.53 (s, 3H), 3.32 (s, 2H), 1.13 (s, 6H). MS-ESI calculated value [M+H] + 296, measured value 296.

實施例39。 Example 39.

1-(2-((1-羥基環丁基)甲氧基)乙基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1- (2 - ((1-hydroxy-cyclobutyl) methoxy) ethyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

Figure 105135042-A0305-02-0120-108
Figure 105135042-A0305-02-0120-108

第一步。 first step.

1-(羥基甲基)環丁醇。 1-(Hydroxymethyl)cyclobutanol.

在25℃條件下，向四氫鋁鋰(1.52g，40.0mmol)的四氫呋喃(30mL)溶液中滴加1-羥基環丁酸(1.16g，10.0mmol)的四氫呋喃(10mL)溶液。反應液加熱至回流，反應1小時。反應液降至25℃，加水(20mL)淬滅，用乙酸乙酯(50mL x 3)萃取，有機相用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到1-(羥基甲基)環丁醇(0.800g，無色油狀物)，產率：80%。 At 25° C., a solution of 1-hydroxycyclobutyric acid (1.16 g, 10.0 mmol) in tetrahydrofuran (10 mL) was added dropwise to a solution of lithium tetrahydroaluminum (1.52 g, 40.0 mmol) in tetrahydrofuran (30 mL). The reaction solution was heated to reflux and reacted for 1 hour. The reaction solution was reduced to 25°C, quenched with water (20 mL), extracted with ethyl acetate (50 mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 1-(hydroxymethyl)cyclobutane Alcohol (0.800g, colorless oil), yield: 80%.

第二步。 The second step.

2-(3，7-二甲基-2，6-二氧-2，3，6，7-四氫-1H-嘌呤-1-基)乙基甲烷磺酸酯。 2-(3,7-Dimethyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)ethylmethanesulfonate.

在0℃向1-(2-羥基乙基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(448mg，2.00mmol)的二氯甲烷(25mL)溶液中加入三乙胺(600mg，6.00mmol)和甲烷磺醯氯(342mg，3.00mmol)。反應液在0℃攪拌0.5小時。加入飽和碳酸氫鈉溶液(30mL)淬滅反應，用二氯甲烷(20mL x 3)萃取。有機相用飽和食鹽水(20mL x 3)洗滌，無水硫酸鈉乾燥，過濾，減壓濃縮得到2-(3，7-二甲基-2，6-二氧-2，3，6，7-四氫-1H-嘌呤-1-基)乙基甲烷磺酸酯(650mg，黃色油狀物)，產率：100%。 In the solution of 1- (2-hydroxyethyl) -3,7-dimethyl -1 H 0 ℃ - purin -2,6 (3 H, 7 H) - dione (448mg, 2.00mmol) in dichloromethane (25mL) Triethylamine (600mg, 6.00mmol) and methanesulfonyl chloride (342mg, 3.00mmol) were added to the solution. The reaction solution was stirred at 0°C for 0.5 hour. The reaction was quenched by adding saturated sodium bicarbonate solution (30 mL) and extracted with dichloromethane (20 mL x 3). The organic phase was washed with saturated brine (20 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 2-(3,7-dimethyl-2,6-dioxo-2,3,6,7- Tetrahydro-1 H -purin-1-yl) ethyl methanesulfonate (650 mg, yellow oil), yield: 100%.

第三步。 third step.

1-(2-((1-羥基環丁基)甲氧基)乙基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1- (2 - ((1-hydroxy-cyclobutyl) methoxy) ethyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

向混合物1-(羥基甲基)環丁醇(102mg，1.00mmol)和2-(3，7-二甲基-2，6-二氧-2，3，6，7-四氫-1H-嘌呤-1-基)乙基甲烷磺酸酯(450mg，1.50mmol)的N，N-二甲基甲醯胺(5mL)溶液中加入碳酸鉀(414mg，3.00mmol)和碘化鉀(16.0mg，0.100mmol)。反應液加熱至60℃攪拌過 夜。然後慢慢降至室溫，加水(20mL)淬滅。混合物用乙酸乙酯(20mL x 3)萃取，有機相用飽和食鹽水(20mL x 3)洗滌，無水硫酸鈉乾燥。過濾，濾液減壓濃縮，用製備高效液相色譜法分離純化得到1-(2-((1-羥基環丁基)甲氧基)乙基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(50.0mg，白色固體)，產率：16%。1H NMR：(400MHz，Methonal-d 4 )δ 7.88(s，1H)，4.57-4.59(m，2H)，4.21-4.24(m，2H)，3.98(s，3H)，3.80(s，2H)，3.54(s，3H)，2.07-1.95(m，4H)，1.52-1.54(m，2H)。MS-ESI計算值[M+H]+ 309，實測值309。 To the mixture 1-(hydroxymethyl)cyclobutanol (102mg, 1.00mmol) and 2-(3,7-dimethyl-2,6-diox-2,3,6,7-tetrahydro-1 H -Purin-1-yl) ethyl methanesulfonate (450mg, 1.50mmol) in N , N -dimethylformamide (5mL) was added potassium carbonate (414mg, 3.00mmol) and potassium iodide (16.0mg, 0.100mmol). The reaction solution was heated to 60°C and stirred overnight. It was then slowly cooled to room temperature and quenched by adding water (20 mL). The mixture was extracted with ethyl acetate (20 mL x 3), the organic phase was washed with saturated brine (20 mL x 3), and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and separated and purified by preparative high performance liquid chromatography to obtain 1-(2-((1-hydroxycyclobutyl)methoxy)ethyl)-3,7-dimethyl-1 H- purine -2,6 (3 H, 7 H) - dione (50.0 mg, white solid), yield: 16%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.88 (s, 1H), 4.57-4.59 (m, 2H), 4.21-4.24 (m, 2H), 3.98 (s, 3H), 3.80 (s, 2H) ), 3.54 (s, 3H), 2.07-1.95 (m, 4H), 1.52-1.54 (m, 2H). MS-ESI calculated value [M+H] + 309, measured value 309.

實施例40。 Example 40.

(S)-1-(2-((2-羥基丙基)氨基)乙基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 (S) -1- (2 - ( (2- hydroxypropyl) amino) ethyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

Figure 105135042-A0305-02-0122-109
Figure 105135042-A0305-02-0122-109

第一步。 first step.

1-(3-氯丙基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮 1-(3-chloropropyl)-3,7-dimethyl-1 H -purine-2,6(3 H ,7 H )-dione

將3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(1.00g，5.56mmol)溶於甲醇(20mL)，加入30%的甲醇鈉(9.64g，49.9mmol)，反應液回流1小時。再加入1-溴-2-氯乙烷(47.2g，299mmol)，反應液繼續攪拌16小時。加入水(30mL)淬滅反應，用二氯甲烷(20mL x 3)萃取，有機相用飽和食鹽水(20mL x 3)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，並用柱層析法分離純化(1：2石油醚/乙酸乙酯)得到1-(3-氯丙基) -3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(230mg，白色固體)，產率：17%。1H NMR：(400MHz，CDCl3)δ7.50(s，1H)，4.36(t，J=6.4Hz，2H)，3.97(s，3H)，3.75(t，J=6.4Hz，2H)，3.56(s，3H). Dissolve 3,7-dimethyl- 1H -purine-2,6(3H,7H)-dione (1.00g, 5.56mmol) in methanol (20mL), add 30% sodium methoxide (9.64g, 49.9 mmol), the reaction solution was refluxed for 1 hour. Then 1-bromo-2-chloroethane (47.2 g, 299 mmol) was added, and the reaction solution was stirred for 16 hours. The reaction was quenched by adding water (30 mL) and extracted with dichloromethane (20 mL x 3). The organic phase was washed with saturated brine (20 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and subjected to column chromatography. purification (1: 2 petroleum ether / ethyl acetate) to give 1- (3-chloropropyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (230 mg, white solid), yield: 17%. 1 H NMR: (400MHz, CDCl 3 ) δ 7.50 (s, 1H), 4.36 (t, J = 6.4 Hz, 2H), 3.97 (s, 3H), 3.75 (t, J = 6.4 Hz, 2H), 3.56(s, 3H).

第二步。 The second step.

(S)-1-(2-((2-羥基丙基)氨基)乙基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。 (S) -1- (2 - ( (2- hydroxypropyl) amino) ethyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione.

在25℃下，向混合物1-(3-氯丙基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(62.4mg，0.826mmol)和(S)-1-氨基丙烷-2-醇(50.0mg，0.207mmol)的乙腈(2mL)溶液中加入碳酸鉀(138mg，1.03mmol)和碘化鉀(86.3mg，0.517mmol)。反應液在90℃攪拌4小時。加入水(10mL)淬滅反應，用乙酸乙酯萃取(20mL x 3)。有機相用飽和食鹽水(20mL x 3)洗滌，無水硫酸鈉乾燥後減壓濃縮。用製備高效液相色譜分離純化得到(S)-1-(2-((2-羥基丙基)氨基)乙基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(10.0mg，白色固體)，產率：17%。1H NMR：(400MHz，Methonal-d 4 )δ 7.86(s，1H)，4.16(t，J=6.4Hz，2H)，3.97(s，3H)，3.84(m，1H)，3.56(s，3H)，2.93(m，2H)，2.64(m，2H)，1.14(d，J=6.4Hz，3H)。MS-ESI計算值[M+H]+ 282，實測值282。 At 25 ℃, the mixture of 1- (3-chloropropyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - dione (62.4mg, 0.826mmol) And ( S )-1-aminopropan-2-ol (50.0 mg, 0.207 mmol) in acetonitrile (2 mL) were added potassium carbonate (138 mg, 1.03 mmol) and potassium iodide (86.3 mg, 0.517 mmol). The reaction solution was stirred at 90°C for 4 hours. The reaction was quenched by adding water (10 mL) and extracted with ethyl acetate (20 mL x 3). The organic phase was washed with saturated brine (20 mL x 3), dried over anhydrous sodium sulfate and concentrated under reduced pressure. Separated and purified by preparative high performance liquid chromatography to obtain ( S )-1-(2-((2-hydroxypropyl)amino)ethyl)-3,7-dimethyl- 1H -purine-2,6(3 H, 7 H) - dione (10.0 mg, white solid), yield: 17%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.86 (s, 1H), 4.16 (t, J = 6.4 Hz, 2H), 3.97 (s, 3H), 3.84 (m, 1H), 3.56 (s, 3H), 2.93 (m, 2H), 2.64 (m, 2H), 1.14 (d, J = 6.4 Hz, 3H). MS-ESI calculated value [M+H] + 282, measured value 282.

實施例41。 Example 41.

Figure 105135042-A0305-02-0123-110
Figure 105135042-A0305-02-0123-110

Figure 105135042-A0305-02-0124-111
Figure 105135042-A0305-02-0124-111

第一步。 first step.

1-(1，4-二氧雜螺[4.5]癸烷-8-基甲基)-7-(二氟甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1-(1,4-Dioxaspiro[4.5]decane-8-ylmethyl)-7-(difluoromethyl)-3-methyl-1 H -purine-2,6(3 H , 7 H )-Diketone.

1-(1，4-二氧雜螺[4.5]癸烷-8-基甲基)-9-(二氟甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1-(1,4-Dioxaspiro[4.5]decane-8-ylmethyl)-9-(difluoromethyl)-3-methyl-1 H -purine-2,6(3 H , 7 H )-Diketone.

將7-(二氟甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮和9-(二氟甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮的混合物(200mg，0.930mmol)溶於N，N-二甲基甲醯胺(20mL)中，反應液於室溫條件下加入1，4-二氧雜螺[4.5]癸烷-8-基甲基甲磺酸酯(245mg，1.10mmol)，碘化鉀(183mg，1.10mmol)和碳酸鉀(303mg，2.20mmol)。反應液加熱到100℃攪拌2小時。反應液中加入乙酸乙酯(30mL)稀釋，有機相用水(20mL x 2)洗滌，無水硫酸鈉乾燥，濃縮得到1-(1，4-二氧雜螺[4.5]癸烷-8-基甲基)-7-(二氟甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮和1-(1，4-二氧雜螺[4.5]癸烷-8-基甲基)-9-(二氟甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮的混合物(234mg，黃色油狀物)，產率：68%。 7- (difluoromethyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione and 9- (difluoromethyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione mixture (200mg, 0.930mmol) was dissolved in N, N - dimethylformamide (20mL), the reaction solution was added at room temperature 1 , 4-Dioxaspiro[4.5]decane-8-yl methanesulfonate (245 mg, 1.10 mmol), potassium iodide (183 mg, 1.10 mmol) and potassium carbonate (303 mg, 2.20 mmol). The reaction solution was heated to 100°C and stirred for 2 hours. The reaction solution was diluted with ethyl acetate (30mL), the organic phase was washed with water (20mL x 2), dried over anhydrous sodium sulfate, and concentrated to obtain 1-(1,4-dioxaspiro[4.5]decane-8-methyl Yl)-7-(difluoromethyl)-3-methyl-1 H -purine-2,6(3 H ,7 H )-dione and 1-(1,4-dioxaspiro[4.5] decan-8-yl-methyl) -9- (difluoromethyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione mixture (234mg, yellow oil物), the yield: 68%.

MS-ESI計算值[M+H]+ 371，實測值371。 MS-ESI calculated value [M+H] + 371, measured value 371.

第二步。 The second step.

7-(二氟甲基)-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮。 7- (difluoromethyl) -3-methyl-1 - ((4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione.

9-(二氟甲基)-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮。 9- (difluoromethyl) -3-methyl-1 - ((4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione.

將1-(1，4-二氧雜螺[4.5]癸烷-8-基甲基)-7-(二氟甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮，1-(1，4-二氧雜螺[4.5]癸烷-8-基甲基)-9-(二氟甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮的混合物(230mg，0.750mmol)溶於四氫呋喃(15mL)中，室溫條件下加入10%鹽酸(5mL)，反應液加熱至50℃攪拌1小時。冷却至室溫，加入乙酸乙酯(20mL)稀釋，有機相用飽和碳酸氫鈉(20mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱層析法分離純化(2：1石油醚/乙酸乙酯，Rf=0.3)得到7-(二氟甲基)-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮，9-(二氟甲基)-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮的混合物(200mg，白色固體)，產率：81%。 The 1-(1,4-dioxaspiro[4.5]decane-8-ylmethyl)-7-(difluoromethyl)-3-methyl-1 H -purine-2,6(3 H , 7 H )-Diketone, 1-(1,4-dioxaspiro[4.5]decane-8-ylmethyl)-9-(difluoromethyl)-3-methyl-1 H -purine -2,6 (3 H, 7 H) - dione mixture (230mg, 0.750mmol) was dissolved in tetrahydrofuran (15mL) was added 10% hydrochloric acid (5mL) at room temperature, the reaction was heated to 50 deg.] C and stirred for 1 hour. Cool to room temperature, add ethyl acetate (20mL) to dilute, the organic phase was washed with saturated sodium bicarbonate (20mL x 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, separated and purified by silica gel column chromatography (2 :1 petroleum ether/ethyl acetate, Rf=0.3) to obtain 7-(difluoromethyl)-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -purine-2,6 (3 H , 7 H )-Diketone, 9-(difluoromethyl)-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -purine-2,6(3 H , 7 H )-diketone mixture (200 mg, white solid), yield: 81%.

MS-ESI計算值[M+H]+ 327，實測值327。 MS-ESI calculated value [M+H] + 327, measured value 327.

第三步。 third step.

7-(二氟甲基)-1-(4-羥基-4-(三氟甲基)環己基)甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 7-(Difluoromethyl)-1-(4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -purine-2,6(3 H , 7 H )-Diketone.

9-(二氟甲基)-1-(4-羥基-4-(三氟甲基)環己基)甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 9-(Difluoromethyl)-1-(4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -purine-2,6(3 H , 7 H )-Diketone.

將7-(二氟甲基)-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮，9-(二氟甲基)-3-甲基-1-((4-氧環己基)甲基)-1H- 嘌呤-2，6(3H，7H)-二酮的混合物(168mg，0.515mmol)溶於四氫呋喃(30mL)中，室溫條件下加入三氟甲基三甲基矽烷(109mg，0.773mmol)和氟化銫(15.7mg，0.103mmol)。反應液在室溫攪拌12小時，加入四丁基氟化銨(50.0mg，0.207mmol)，室溫攪拌30分鐘後加入乙酸乙酯(20mL)稀釋，有機相用飽和碳酸氫鈉(20mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮用高效液相色譜純化得7-(二氟甲基)-1-(4-羥基-4-(三氟甲基)環己基)甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(54mg，白色固體)，產率：23%，1H NMR：(400MHz，Methanol-d 4)δ8.46(s，1H)，7.89-7.74(m，1H)，406(d，J=7.2Hz，2H)，3.59(s，3H)，2.19-2.17(m，1H)，2.05-1.99(m，2H)，1.88-1.81(m，2H)，1.61-1.58(m，2H)，1.51-1.47(m，2H)。MS-ESI計算值[M+H]+ 397，實測值397。 7- (difluoromethyl) -3-methyl-1 - ((4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione, 9- (difluoromethyl) -3-methyl-1 - ((4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione mixture (168mg, 0.515 mmol) was dissolved in tetrahydrofuran (30 mL), and trifluoromethyltrimethylsilane (109 mg, 0.773 mmol) and cesium fluoride (15.7 mg, 0.103 mmol) were added at room temperature. The reaction solution was stirred at room temperature for 12 hours, tetrabutylammonium fluoride (50.0mg, 0.207mmol) was added, stirred at room temperature for 30 minutes and then diluted with ethyl acetate (20mL). The organic phase was diluted with saturated sodium bicarbonate (20mL x 2 ) Wash, dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify by HPLC to obtain 7-(difluoromethyl)-1-(4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl )-3-Methyl-1 H -purine-2,6(3 H , 7 H )-dione (54 mg, white solid), yield: 23%, 1 H NMR: (400MHz, Methanol- d 4 ) δ8.46 (s, 1H), 7.89-7.74 (m, 1H), 406 (d, J = 7.2Hz, 2H), 3.59 (s, 3H), 2.19-2.17 (m, 1H), 2.05-1.99 ( m, 2H), 1.88-1.81 (m, 2H), 1.61-1.58 (m, 2H), 1.51-1.47 (m, 2H). MS-ESI calculated value [M+H] + 397, measured value 397.

9-(二氟甲基)-1-(4-羥基-4-(三氟甲基)環己基)甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(12mg，白色固體)，產率：10%。1H NMR：(400MHz，Methanol-d 4 )δ8.49(s，1H)，7.93-7.89(m，1H)，4.07(d，J=7.2Hz，2H)，3.59(s，3H)，2.20-2.19(m，1H)，2.05-1.99(m，2H)，1.88-1.85(m，2H)，1.61-1.58(m，2H)，1.51-1.48(m，2H)。MS-ESI計算值[M+H]+ 397，實測值397。 9-(Difluoromethyl)-1-(4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -purine-2,6(3 H , 7 H )-Diketone (12 mg, white solid), yield: 10%. 1 H NMR: (400MHz, Methanol- d 4 )δ8.49(s, 1H), 7.93-7.89(m, 1H), 4.07(d, J = 7.2Hz, 2H), 3.59(s, 3H), 2.20 -2.19 (m, 1H), 2.05-1.99 (m, 2H), 1.88-1.85 (m, 2H), 1.61-1.58 (m, 2H), 1.51-1.48 (m, 2H). MS-ESI calculated value [M+H] + 397, measured value 397.

實施例42。 Example 42.

7-乙基-1-(5-乙基-5-羥基庚基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 7-ethyl-1- (5-ethyl-5-hydroxy-hept-yl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione.

Figure 105135042-A0305-02-0126-112
Figure 105135042-A0305-02-0126-112
Figure 105135042-A0305-02-0127-113
Figure 105135042-A0305-02-0127-113

第一步。 first step.

7-乙基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 7-ethyl-3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione.

將3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(500mg，3.00mmol)，碳酸鉀(414mg，3.00mmol)和碘化鉀(4.0mg，0.300mmol)溶解於N，N-二甲基甲醯胺(15mL)中。反應液加熱到80℃反應半小時。加入碘乙烷(470mg，4.50mmol)。繼續反應5小時。把反應液倒入氫氧化鈉水溶液(50mL)中淬滅反應，用乙酸乙酯(20mL x 3)萃取。水相用1N稀鹽酸(10mL)調節酸鹼度至pH值至7，過濾，濾餅乾燥後得到7-乙基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(500mg，淡黃色固體)，產率：86%。1H NMR：(400MHz，DMSO-d 6)：δ8.05(s，1H)，4.25-4.19(m，2H)，3.34(s，3H)，1.37(t，J=7.2Hz，3H)。MS-ESI計算值[M+H]+ 195，實測值195. 3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (500mg, 3.00mmol), potassium carbonate (414mg, 3.00mmol) and potassium iodide (4.0mg, 0.300mmol) was dissolved in N , N -dimethylformamide (15 mL). The reaction solution was heated to 80°C for half an hour. Add iodoethane (470 mg, 4.50 mmol). Continue the reaction for 5 hours. The reaction solution was poured into an aqueous sodium hydroxide solution (50 mL) to quench the reaction, and extracted with ethyl acetate (20 mL x 3). Adjust the pH of the aqueous phase to pH 7 with 1N dilute hydrochloric acid (10 mL), filter, and dry the filter cake to obtain 7-ethyl-3-methyl-1 H -purine-2,6(3 H ,7 H )- Dione (500 mg, pale yellow solid), yield: 86%. 1 H NMR: (400 MHz, DMSO- d 6 ): δ 8.05 (s, 1H), 4.25-4.19 (m, 2H), 3.34 (s, 3H), 1.37 (t, J = 7.2 Hz, 3H). MS-ESI calculated value [M+H] + 195, measured value 195.

第二步。 The second step.

乙基5-(7-乙基-3-甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)戊酸乙酯。 Ethyl 5-(7-ethyl-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)pentanoic acid ethyl ester.

將7-乙基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(0.300g，1.55mmol)，溴戊酸乙酯(480mg，2.32mmol)，碳酸鉀(430mg，3.10mmol)和碘化鉀(26.0mg，0.155mmol)溶解於N，N-二甲基甲醯胺(4mL)中。反應液加熱到110℃反應2小時。把反應液倒入水中(20mL)淬滅反應，用乙酸乙酯萃取(20mL x 3)。合併有機相，用無水硫酸鈉乾燥，過濾，減 壓濃縮得到乙基5-(7-乙基-3-甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)戊酸乙酯(320mg，黃色固體)，產率：62%。MS-ESI計算值[M+H]+ 323，實測值323。 7-ethyl-3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (0.300g, 1.55mmol), bromo-pentanoate (480mg, 2.32mmol), carbonate Potassium (430 mg, 3.10 mmol) and potassium iodide (26.0 mg, 0.155 mmol) were dissolved in N , N -dimethylformamide (4 mL). The reaction solution was heated to 110°C for 2 hours. The reaction solution was poured into water (20 mL) to quench the reaction, and extracted with ethyl acetate (20 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain ethyl 5-(7-ethyl-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro- 1 H -purin-1-yl)ethyl valerate (320 mg, yellow solid), yield: 62%. MS-ESI calculated value [M+H] + 323, measured value 323.

第三步。 third step.

7-乙基-1-(5-乙基-5-羥基庚基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 7-ethyl-1- (5-ethyl-5-hydroxy-hept-yl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione.

將乙基5-(7-乙基-3-甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)戊酸乙酯(0.100g，0.310mmol)溶解於無水四氫呋喃(10mL)中，於-78℃條件下慢慢滴加乙基溴化鎂(3M四氫呋喃溶液，0.62mL，1.86mmol)。反應液在-78℃反應0.5小時，緩慢升至0℃反應0.5小時。反應完全後，反應液倒入水(20mL)中，用乙酸乙酯萃取(30mL x 3)。有機相用無水硫酸鈉乾燥，過濾，減壓濃縮，用矽膠色譜柱純化得到7-乙基-1-(5-乙基-5-羥基庚基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(30.0mg，無色油狀物)，產率：30%。 Ethyl 5-(7-ethyl-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)pentanoic acid ethyl ester (0.100 g, 0.310mmol) was dissolved in anhydrous tetrahydrofuran (10mL), and ethylmagnesium bromide (3M tetrahydrofuran solution, 0.62mL, 1.86mmol) was slowly added dropwise at -78°C. The reaction solution was reacted at -78°C for 0.5 hours, and slowly raised to 0°C for 0.5 hours. After the reaction was completed, the reaction solution was poured into water (20 mL), and extracted with ethyl acetate (30 mL x 3). The organic phase was dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified with a silica gel column to obtain 7-ethyl-1-(5-ethyl-5-hydroxyheptyl)-3-methyl-1 H -purine- 2,6 (3 H, 7 H) - dione (30.0 mg, colorless oil). yield: 30%.

1H NMR：(400MHz，CDCl3)：δ7.56(s，1H)，4.37-4.32(m，2H)，4.05(t，J=7.2Hz，2H)，3.60(s，3H)，1.68-1.37(m，13H)，0.86(t，J=7.2Hz，6H)。MS-ESI計算值[M+H]+ 337，實測值337。 1 H NMR: (400MHz, CDCl 3 ): δ 7.56 (s, 1H), 4.37-4.32 (m, 2H), 4.05 (t, J = 7.2 Hz, 2H), 3.60 (s, 3H), 1.68- 1.37 (m, 13H), 0.86 (t, J = 7.2 Hz, 6H). MS-ESI calculated value [M+H] + 337, measured value 337.

實施例43。 Example 43.

7-乙基-3-甲基-1-(6，6，6-三氟-5-羥基-5-甲基己基)-1H-嘌呤-2，6(3H，7H)-二酮。 7-Ethyl-3-methyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)-1 H -purine-2,6(3 H ,7 H )-di ketone.

Figure 105135042-A0305-02-0128-115
Figure 105135042-A0305-02-0128-115
Figure 105135042-A0305-02-0129-117
Figure 105135042-A0305-02-0129-117

第一步。 first step.

7-乙基-3-甲基-1-(5-氧代己基)-1H-嘌呤-2，6(3H，7H)-二酮 7-Ethyl-3-methyl-1-(5-oxohexyl)-1 H -purine-2,6(3 H ,7 H )-dione

將7-乙基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(0.100g，0.515mmol)，6-氯-2-戊酮(90.0mg，0.670mmol)，碳酸鉀(140mg，1.03mmol)和碘化鉀(8.5mg，0.0155mmol)溶解於DMF(2mL)中，加熱到110℃反應兩小時。把反應倒入水中，用乙酸乙酯萃取(20mL x 3)。合併有機相，乾燥，過濾，濃縮並用三級丁基甲醚洗滌，乾燥固體得目標化合物7-乙基-3-甲基-1-(5-氧代己基)-1H-嘌呤-2，6(3H，7H)-二酮(100mg，白色固體)，產率：70%。MS-ESI計算值[M+H]+293，實測值293。 7-ethyl-3-methyl -1H- purine -2,6 (3 H, 7 H) - dione (0.100g, 0.515mmol), 6- chloro-2-pentanone (90.0mg, 0.670mmol ), potassium carbonate (140mg, 1.03mmol) and potassium iodide (8.5mg, 0.0155mmol) were dissolved in DMF (2mL) and heated to 110°C for two hours. The reaction was poured into water and extracted with ethyl acetate (20 mL x 3). The organic phases were combined, dried, filtered, concentrated and washed with tertiary butyl methyl ether. The solid was dried to obtain the target compound 7-ethyl-3-methyl-1-(5-oxohexyl)-1 H -purine-2,6( 3 H , 7 H )-dione (100 mg, white solid), yield: 70%. MS-ESI calculated value [M+H] + 293, measured value 293.

第二步。 The second step.

7-乙基-3-甲基-1-(6，6，6-三氟-5-羥基-5-甲基己基)-1H-嘌呤-2，6(3H，7H)-二酮。 7-Ethyl-3-methyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)-1 H -purine-2,6(3 H ,7 H )-di ketone.

將7-乙基-3-甲基-1-(5-氧代己基)-1H-嘌呤-2，6(3H，7H)-二酮(100mg，0.340mmol)溶解於1毫升的四氫呋喃中，依次加入三氟甲基三甲基矽烷(53.0mg，0.370mmol)和氟化銫(10.0mg，0.0340mmol)，於30℃反應3小時。把反應液倒入的稀鹽酸(10%，10mL)中，攪拌半小時。用乙酸乙酯萃取(20mL x 3)。合併有機相，乾燥，濃縮，殘渣用製備色譜柱純化的目標化合物7-乙基-3-甲基-1-(6，6，6-三氟-5-羥基-5-甲基 己基)-1H-嘌呤-2，6(3H，7H)-二酮(20.0mg，白色固體)，產率：79%。1H NMR：(400MHz，CDCl3)：δ7.95(s，1H)，4.39-4.33(m，2H)，4.04-4.00(m，2H)，3.53(s，3H)，1.71-1.64(m，4H)，1.50-1.46(m，5H)，1.28(s，3H)。MS-ESI計算值[M+H]+363，實測值363。 7-ethyl-3-methyl-1- (5-oxo-hexyl) -1 H - purine -2,6 (3 H, 7 H) - dione (100mg, 0.340mmol) was dissolved in 1 ml of To tetrahydrofuran, add trifluoromethyltrimethylsilane (53.0mg, 0.370mmol) and cesium fluoride (10.0mg, 0.0340mmol) in sequence, and react at 30°C for 3 hours. Pour the reaction solution into diluted hydrochloric acid (10%, 10 mL) and stir for half an hour. Extract with ethyl acetate (20 mL x 3). The organic phases were combined, dried, concentrated, and the residue was purified with a preparative column to purify the target compound 7-ethyl-3-methyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)- 1 H - purine -2,6 (3 H, 7 H) - dione (20.0 mg of, white solid), yield: 79%. 1 H NMR: (400MHz, CDCl 3 ): δ 7.95 (s, 1H), 4.39-4.33 (m, 2H), 4.04-4.00 (m, 2H), 3.53 (s, 3H), 1.71-1.64 (m , 4H), 1.50-1.46 (m, 5H), 1.28 (s, 3H). MS-ESI calculated value [M+H] + 363, measured value 363.

實施例44。 Example 44.

1-((4-羥基-4-(三氟甲基)環己基)甲基)-3-甲基-7-(2，2，2-三氟乙基)-1H-嘌呤-2，6(3H，7H)-二酮。 1-((4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-7-(2,2,2-trifluoroethyl)-1 H -purine-2, 6(3 H , 7 H )-dione.

Figure 105135042-A0305-02-0130-119
Figure 105135042-A0305-02-0130-119

第一步。 first step.

1-(1，4-二氧雜螺[4，5]癸烷-8-基甲基)-3-甲基-7-(2，2，2-三氟乙基)-1H-嘌呤-2，6(3H，7H)-二酮 1-(1,4-Dioxaspiro[4,5]decane-8-ylmethyl)-3-methyl-7-(2,2,2-trifluoroethyl)-1 H -purine -2,6(3 H ，7 H )-dione

將1，4-二氧雜螺[4，5]癸烷-8-基甲基甲磺酸酯(200mg，0.800mmol)，3-甲基-7-(2，2，2-三氟乙基)-1H-嘌呤-2，6(3H，7H)-二酮(200g，0.800mmol)及碳酸鉀(334mg，2.42mmol)，碘化鉀(14.0mg，0.0800mmol)溶於N，N-二甲基甲醯胺(3mL)中，反應液加熱至130℃，攪拌3.5小時。反應液直接過濾，濾液減壓濃縮，得到粗產品1-(1，4-二氧 雜螺[4，5]癸烷-8-基甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮。MS-ESI計算值[M+H]+ 403，實測值403。 1,4-Dioxaspiro[4,5]decane-8-yl methyl methanesulfonate (200mg, 0.800mmol), 3-methyl-7-(2,2,2-trifluoroethane) yl) -1 H - purine -2,6 (3 H, 7 H) - dione (200g, 0.800mmol) and potassium carbonate (334mg, 2.42mmol), potassium iodide (14.0mg, 0.0800mmol) was dissolved in N, N -In dimethylformamide (3 mL), the reaction solution was heated to 130°C and stirred for 3.5 hours. The reaction solution was directly filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product 1-(1,4-dioxaspiro[4,5]decane-8-ylmethyl)-3,7-dimethyl-1 H- purine -2,6 (3 H, 7 H) - dione. MS-ESI calculated value [M+H] + 403, measured value 403.

第二步。 The second step.

3-甲基-1-((4-氧代環己基)甲基)-7-(2，2，2-三氟乙基)-1H-嘌呤-2，6(3H，7H)-二酮。 3-methyl-1-((4-oxocyclohexyl)methyl)-7-(2,2,2-trifluoroethyl)-1 H -purine-2,6(3 H , 7 H ) -Diketone.

將1-(1，4-二氧雜螺[4，5]癸烷-8-基甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(2.50g，6.00mmol)溶於丙酮(18mL)中，加入鹽酸水溶液(4N，2.5mL)。反應在30℃攪拌過夜，加入水(50mL)，用乙酸乙酯萃取(20mL x 3)。合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮用矽膠柱色譜法純化(1：3石油醚/乙酸乙酯，Rf=0.3)得到產物3-甲基-1-((4-氧代環己基)甲基)-7-(2，2，2-三氟乙基)-1H-嘌呤-2，6(3H，7H)-二酮(220mg，白色固體)，產率：11%。1H NMR：(400MHz，CDCl3)δ7.68(s，1H)，5.08-4.99(m，2H)，4.00(d，J=7.0Hz，2H)，3.61(s，3H)，2.46-2.24(m，5H)，2.04-1.96(m，2H)，1.63-1.56(m，2H)。MS-ESI計算值[M+H]+ 359，實測值359。 1- (1,4-dioxaspiro [4,5] decan-8-yl-methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) -Dione (2.50 g, 6.00 mmol) was dissolved in acetone (18 mL), and aqueous hydrochloric acid solution (4N, 2.5 mL) was added. The reaction was stirred at 30°C overnight, water (50 mL) was added, and it was extracted with ethyl acetate (20 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (1:3 petroleum ether/ethyl acetate, Rf=0.3) to obtain the product 3-methyl-1-((4- Oxocyclohexyl) methyl)-7-(2,2,2-trifluoroethyl)-1 H -purine-2,6(3 H ,7 H )-dione (220mg, white solid), Rate: 11%. 1 H NMR: (400MHz, CDCl 3 ) δ 7.68 (s, 1H), 5.08-4.99 (m, 2H), 4.00 (d, J = 7.0 Hz, 2H), 3.61 (s, 3H), 2.46-2.24 (m, 5H), 2.04-1.96 (m, 2H), 1.63-1.56 (m, 2H). MS-ESI calculated value [M+H] + 359, measured value 359.

第三步。 third step.

1-((4-羥基-4-(三氟甲基)環己基)甲基)-3-甲基-7-(2，2，2-三氟乙基)-1H-嘌呤-2，6(3H，7H)-二酮。 1-((4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-7-(2,2,2-trifluoroethyl)-1 H -purine-2, 6(3 H , 7 H )-dione.

將3-甲基-1-((4-氧代環己基)甲基)-7-(2，2，2-三氟乙基)-1H-嘌呤-2，6(3H，7H)-二酮(128mg，0.360mmol)及氟化銫(6.0mg，0.0360mmol)溶於四氫呋喃(3mL)中，在氮氣保護下緩慢加入三氟甲基三甲基矽烷(77.0mg，0.540mmol)。反應液在30℃下攪拌3小時。冷却至室溫，加入4N鹽酸水溶液(2.5mL)，在25℃攪拌半小時，調節pH值至7，加水稀釋，用乙酸乙酯萃取(20mL x 3)。合併有機相，用無水硫酸鈉乾 燥，過濾，減壓濃縮，用製備高效液相色譜純化得產物1-((4-羥基-4-(三氟甲基)環己基)甲基)-3-甲基-7-(2，2，2-三氟乙基)-1H-嘌呤-2，6(3H，7H)-二酮(14.0mg，白色固體)，產率：10%。1H NMR：(400MHz，CDCl3)δ8.09(s，1H)，5.27-5.20(m，2H)，4.08-3.91(m，2H)，3.58(s，3H)，2.07-1.98(m，2H)，1.89-1.80(m，2H)，1.62-1.46(m，5H)。MS-ESI計算值[M+H]+ 429，實測值429。 3-methyl-1 - ((4-oxo-cyclohexyl) methyl) -7- (2,2,2-trifluoroethyl) -1 H - purine -2,6 (3 H, 7 H )-Diketone (128mg, 0.360mmol) and cesium fluoride (6.0mg, 0.0360mmol) were dissolved in tetrahydrofuran (3mL), and trifluoromethyltrimethylsilane (77.0mg, 0.540mmol) was slowly added under the protection of nitrogen. . The reaction solution was stirred at 30°C for 3 hours. Cool to room temperature, add 4N aqueous hydrochloric acid (2.5 mL), stir at 25° C. for half an hour, adjust the pH to 7, dilute with water, and extract with ethyl acetate (20 mL x 3). The organic phases were combined, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by preparative high performance liquid chromatography to obtain the product 1-((4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3- methyl-7- (2,2,2-trifluoroethyl) -1 H - purine -2,6 (3 H, 7 H) - dione (14.0 mg, white solid), yield: 10%. 1 H NMR: (400MHz, CDCl 3 ) δ 8.09 (s, 1H), 5.27-5.20 (m, 2H), 4.08-3.91 (m, 2H), 3.58 (s, 3H), 2.07-1.98 (m, 2H), 1.89-1.80 (m, 2H), 1.62-1.46 (m, 5H). MS-ESI calculated value [M+H] + 429, measured value 429.

實施例45。 Example 45.

Figure 105135042-A0305-02-0132-120
Figure 105135042-A0305-02-0132-120

1-((4-羥基-4-(三氟甲基)環己基)甲基)-3-甲基-7-(2，2，2-三氟乙基)-1H-嘌呤-2，6(3H，7H)-二酮(500mg，1.17mmol)，通過製備SFC來分離得到兩個異構體。分離條件：色譜柱：AD 250mm x 30mm，10um流動相：A：超臨界二氧化碳，B：乙醇(0.05%氨水)，A：B=550：45流速：80mL/min波長：220nm。 1-((4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-7-(2,2,2-trifluoroethyl)-1 H -purine-2, 6 (3 H, 7 H) - dione (500mg, 1.17mmol), separated by preparative SFC to give two isomers. Separation conditions: Chromatographic column: AD 250mm x 30mm, 10um mobile phase: A: supercritical carbon dioxide, B: ethanol (0.05% ammonia), A: B=550:45 Flow rate: 80mL/min Wavelength: 220nm.

產物1(異構體1，第一個峰)(300mg，白色固體)，產率：90%。1H NMR：(400MHz，DMSO-d 6)δ8.23(s，1H)，5.64(s，1H)，5.31-5.24(m，2H)，3.89(d，J=3.6Hz，2H)，3.43(s，3H)，2.06-2.05(m，1H)，1.87-1.81(m，2H)，1.73-1.61(m，2H)，1.49-1.45(m，2H)，1.33-1.31(m，2H).MS ESI calc’d.[M+H]+ 429，found 429。 Product 1 (isomer 1, first peak) (300 mg, white solid), yield: 90%. 1 H NMR: (400MHz, DMSO- d 6 ) δ 8.23 (s, 1H), 5.64 (s, 1H), 5.31-5.24 (m, 2H), 3.89 (d, J = 3.6 Hz, 2H), 3.43 (s, 3H), 2.06-2.05 (m, 1H), 1.87-1.81 (m, 2H), 1.73-1.61 (m, 2H), 1.49-1.45 (m, 2H), 1.33-1.31 (m, 2H) .MS ESI calc'd.[M+H] + 429, found 429.

產物2(異構體2，第二個峰)(150mg，白色固體)，產率90%。1H NMR：(400MHz，DMSO-d 6)δ8.22(s，1H)，5.63(s，1H)，5.29-5.23(m，2H)，3.74(d，J=3.6Hz，2H)，3.42(s，3H)，1.68-1.66(m，3H)，1.45-1.31(m，6H).MS ESI calc’d.[M+H]+ 429，found 429。 Product 2 (isomer 2, second peak) (150 mg, white solid), yield 90%. 1 H NMR: (400MHz, DMSO- d 6 ) δ 8.22 (s, 1H), 5.63 (s, 1H), 5.29-5.23 (m, 2H), 3.74 (d, J = 3.6 Hz, 2H), 3.42 (s, 3H), 1.68-1.66 (m, 3H), 1.45-1.31 (m, 6H). MS ESI calc'd. [M+H] + 429, found 429.

實施例46。 Example 46.

Figure 105135042-A0305-02-0133-122
Figure 105135042-A0305-02-0133-122

第一步。 first step.

1-(1，4-二氧雜螺[4，5]癸烷-8-基甲基)-3-甲基-7-(2，2，2-三氟乙基)-1H-嘌呤-2，6-(3H，7H)-二酮。 1-(1,4-Dioxaspiro[4,5]decane-8-ylmethyl)-3-methyl-7-(2,2,2-trifluoroethyl)-1 H -purine -2,6-(3 H ,7 H )-dione.

將1，4-二氧雜螺[4，5]癸烷-8-基甲基甲磺酸酯(603mg，2.41mmol)，3-甲基-7-(2，2，2-三氟乙基)-1H-嘌呤-2，6-(3H，7H)-二酮(500mg，2.01mmol)及碘化鉀(33.3mg，0.201mmol)溶於N，N-二甲基甲醯胺(8mL)中，加入碳酸鉀(555mg，4.02mmol)，反應130℃加熱回流4小時。反應液冷却至室溫，過濾，濾液減壓濃縮，得到的粗產品1-(1，4-二氧雜螺[4，5]癸烷-8-基甲基)-3-甲基-7-(2，2，2-三氟乙基)-1H- 嘌呤-2，6-(3H，7H)-二酮(980mg，黃色油狀)。MS-ESI計算值[M+H]+ 403，實測值403。 1,4-Dioxaspiro[4,5]decane-8-yl methanesulfonate (603mg, 2.41mmol), 3-methyl-7-(2,2,2-trifluoroethane) yl) -1 H - purine -2,6- (3 H, 7 H) - dione (500mg, 2.01mmol) and potassium iodide (33.3mg, 0.201mmol) was dissolved in N, N - dimethylformamide ( 8mL), potassium carbonate (555mg, 4.02mmol) was added, and the reaction was heated to reflux at 130°C for 4 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product 1-(1,4-dioxaspiro[4,5]decane-8-ylmethyl)-3-methyl-7 - (2,2,2-trifluoroethyl) -1 H - purine -2,6- (3 H, 7 H) - dione (980mg, yellow oil). MS-ESI calculated value [M+H] + 403, measured value 403.

第二步。 The second step.

3-甲基-1-((4-氧代環己基)甲基)-7-(2，2，2-三氟乙基)-1H-嘌呤-2，6-(3H，7H)-二酮。 3-methyl-1-((4-oxocyclohexyl)methyl)-7-(2,2,2-trifluoroethyl)-1 H -purine-2,6-(3 H ,7 H )-Diketone.

將1-(1，4-二氧雜螺[4，5]癸烷-8-基甲基)-3-甲基-7-(2，2，2-三氟乙基)-1H-嘌呤-2，6-(3H，7H)-二酮(980mg，1.51mmol)溶於丙酮(8mL)中，加入4N鹽酸水溶液(2mL)。反應室溫攪拌過夜，加入水(20mL)，用乙酸乙酯(30mL x 3)萃取，有機相用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到的產品用矽膠柱色譜法純化(1：1石油醚/乙酸乙酯，Rf=0.3)得到產物3-甲基-1-((4-氧代環己基)甲基)-7-(2，2，2-三氟乙基)-1H-嘌呤-2，6-(3H，7H)-二酮(78.0mg，黃色固體)，產率：15%。MS-ESI計算值[M+H]+ 359，實測值359。 The 1-(1,4-dioxaspiro[4,5]decane-8-ylmethyl)-3-methyl-7-(2,2,2-trifluoroethyl)-1 H- purine -2,6- (3 H, 7 H) - dione (980mg, 1.51mmol) was dissolved in acetone (8 mL) was added 4N aqueous hydrochloric acid (2mL). The reaction was stirred overnight at room temperature, water (20 mL) was added, and the mixture was extracted with ethyl acetate (30 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product obtained was purified by silica gel column chromatography (1: 1 Petroleum ether/ethyl acetate, Rf=0.3) to obtain the product 3-methyl-1-((4-oxocyclohexyl)methyl)-7-(2,2,2-trifluoroethyl)-1 H - purine -2,6- (3 H, 7 H) - dione (78.0 mg, yellow solid), yield: 15%. MS-ESI calculated value [M+H] + 359, measured value 359.

將3-甲基-1-((4-氧代環己基)甲基)-7-(2，2，2-三氟乙基)-1H-嘌呤-2，6-(3H，7H)-二酮(66.0mg，0.184mmol)溶於四氫呋喃(2mL)，-78℃時在氮氣保護下緩慢加入甲基格氏試劑(3M乙醚溶液，0.184mL，0.552mmol)，-78℃攪拌半小時，接著0℃反應2小時。加入水(10mL)，用乙酸乙酯(30mL x 3)萃取，有機相用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到粗產品用製備高效液相色譜純化得產物1(8.00mg，白色固體)，產率：12%，1H NMR：(400MHz，Methonal-d 4)δ8.08(s，1H)，5.27-5.19(m，2H)，3.92(d，J=7.2Hz，2H)，3.58(s，3H)，1.71-1.62(m，4H)，1.46-1.38(m，2 H)，1.32-1.18(m，6H)。MS-ESI計算值[M+H-H2O]+ 357，實測值357。 3-methyl-1 - ((4-oxo-cyclohexyl) methyl) -7- (2,2,2-trifluoroethyl) -1 H - purine -2,6- (3 H, 7 H )-diketone (66.0mg, 0.184mmol) dissolved in tetrahydrofuran (2mL), slowly add methyl Grignard reagent (3M ether solution, 0.184mL, 0.552mmol) at -78℃ under nitrogen protection, and stir at -78℃ Half an hour, followed by reaction at 0°C for 2 hours. Water (10mL) was added, extracted with ethyl acetate (30mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. Purified by preparative high performance liquid chromatography to obtain product 1 (8.00mg, white Solid), yield: 12%, 1 H NMR: (400MHz, Methonal- d 4 )δ8.08 (s, 1H), 5.27-5.19 (m, 2H), 3.92 (d, J = 7.2Hz, 2H) , 3.58 (s, 3H), 1.71-1.62 (m, 4H), 1.46-1.38 (m, 2 H), 1.32-1.18 (m, 6H). MS-ESI calculated value [M+HH 2 O] + 357, measured value 357.

產物2(12.0mg，白色固體)(異構體2，第二個峰)，產率：17%。1H NMR：(400MHz，Methonal-d4)δ8.08(s，1H)，5.27-5.21(m，2H)，3.91(d，J=7.2Hz，2H)，3.57(s，3H)，1.69-1.66(m，2H)，1.49-1.44(m，3H)，1.37-1.28(m，4H)，1.17(s，3H)。MS-ESI計算值[M+H-H2O]+ 357，實測值357。 Product 2 (12.0 mg, white solid) (isomer 2, second peak), yield: 17%. 1 H NMR: (400MHz, Methonal-d4) δ 8.08 (s, 1H), 5.27-5.21 (m, 2H), 3.91 (d, J = 7.2 Hz, 2H), 3.57 (s, 3H), 1.69- 1.66 (m, 2H), 1.49-1.44 (m, 3H), 1.37-1.28 (m, 4H), 1.17 (s, 3H). MS-ESI calculated value [M+HH 2 O] + 357, measured value 357.

實施例47。 Example 47.

7-環丙基-3-甲基-1-(6，6，6-三氟-5-羥基-5-甲基己基)-1H-嘌呤-2，6(3H，7H)-二酮。 7-Cyclopropyl-3-methyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)-1 H -purine-2,6(3 H ,7 H )- Diketone.

Figure 105135042-A0305-02-0135-125
Figure 105135042-A0305-02-0135-125

第一步。 first step.

6-氨基-5-溴-1-甲基嘧啶-2，4(1H，3H)-二酮。 6-Amino-5-bromo-1-methylpyrimidine-2,4( 1H , 3H )-dione.

混合物6-氨基-1-甲基嘧啶-2，4(1H，3H)-二酮(5.46g，40.0mmol)和溴代丁二醯亞胺(7.56g，42.0mmol)的乙腈(100mL)溶液在氮氣保護加熱回流1.5小時。反應液冷却到室溫，過濾，除去溶劑，所得固體用水(20mL)洗滌，乾燥得到6-氨基-5-溴-1-甲基嘧啶-2，4(1H，3H)-二酮(8.6g，白色固體)，產率：98%。1H NMR：(400MHz，DMSO-d6)δ10.90(s，1H)，7.04(s，2H)，3.28(s，3H)。 Mixture of 6-amino-1-methylpyrimidine-2,4( 1H , 3H )-dione (5.46g, 40.0mmol) and bromosuccinimide (7.56g, 42.0mmol) in acetonitrile (100mL ) The solution was heated to reflux for 1.5 hours under nitrogen protection. The reaction solution was cooled to room temperature, filtered, and the solvent was removed. The obtained solid was washed with water (20 mL) and dried to obtain 6-amino-5-bromo-1-methylpyrimidine-2,4(1 H , 3 H )-dione ( 8.6g, white solid), yield: 98%. 1 H NMR: (400MHz, DMSO- d6 ) δ 10.90 (s, 1H), 7.04 (s, 2H), 3.28 (s, 3H).

第二步。 The second step.

6-氨基-5-(環丙基胺)-1-甲基嘧啶-2，4(1H，3H)-二酮。 6-Amino-5-(cyclopropylamine)-1-methylpyrimidine-2,4( 1H , 3H )-dione.

6-氨基-5-溴-1-甲基嘧啶-2，4(1H，3H)-二酮(2.19g，10.0mmol)溶解於環丙胺(20mL)和水(5mL)的混合溶劑中。反應液加熱回流5小時。反應液過濾除去溶劑，得到粗產品6-氨基-5-(環丙基胺)-1-甲基嘧啶-2，4(1H，3H)-二酮直接用於下一步反應。 6-Amino-5-bromo-1-methylpyrimidine-2,4( 1H , 3H )-dione (2.19g, 10.0mmol) was dissolved in a mixed solvent of cyclopropylamine (20mL) and water (5mL) . The reaction solution was heated to reflux for 5 hours. The reaction solution was filtered to remove the solvent, and the crude product 6-amino-5-(cyclopropylamine)-1-methylpyrimidine-2,4(1 H , 3 H )-dione was directly used in the next reaction.

第三步。 third step.

7-環丙基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 7-cyclopropyl-3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione.

在氮氣保護下將6-氨基-5-(環丙基胺)-1-甲基嘧啶-2，4(1H，3H)-二酮(1.96g，10.0mmol)，原甲酸三甲酯(2.12g，20.0mmol)和對甲基苯磺酸(86.0mg，0.500mmol)溶解於的無水N，N-二甲基甲醯胺(20mL)中。反應液加熱到100℃反應過夜。反應液過濾，除去溶劑，得到粗產品7-環丙基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮直接用於下一步反應。 Under nitrogen protection, 6-amino-5-(cyclopropylamine)-1-methylpyrimidine-2,4(1 H , 3 H )-dione (1.96g, 10.0mmol), trimethyl orthoformate (2.12g, 20.0mmol) and p-toluenesulfonic acid (86.0mg, 0.500mmol) were dissolved in anhydrous N , N -dimethylformamide (20mL). The reaction solution was heated to 100°C and reacted overnight. The reaction was filtered, the solvent was removed to give crude product 7-cyclopropyl-3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione was used directly in the next reaction.

第四步。 the fourth step.

7-環丙基-3-甲基-1-(5-氧代己烷)-1H-嘌呤-2，6(3H，7H)-二酮。 7-cyclopropyl-3-methyl-1- (5-oxo-hexane) -1 H - purine -2,6 (3 H, 7 H) - dione.

在氮氣保護下將7-環丙基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(100mg，0.480mmol)，6-氯己烷-2-酮(97.0mg，0.730mmol)和碳酸鉀(132mg，0.960mmol)N，N-二甲基甲醯胺(5mL)中。反應液加熱到120℃反應3小時。待反應冷却至室溫後用水(20mL)和乙酸乙酯(10mL)稀釋，乙酸乙酯萃取(30mL x 2)，有機相用無水硫酸鈉乾燥，過濾，濃縮得到粗產品7-環丙基-3-甲基-1-(5-氧代己烷)-1H-嘌呤-2，6(3H，7H)-二酮直接用於下一步反應。MS-ESI計算值[M+H]+ 305，實測值305。 Under the protection of nitrogen, the 7-cyclopropyl-3-methyl-1 H -purine-2,6(3 H , 7 H )-dione (100mg, 0.480mmol), 6-chlorohexane-2-one (97.0 mg, 0.730 mmol) and potassium carbonate (132 mg, 0.960 mmol) N , N -dimethylformamide (5 mL). The reaction solution was heated to 120°C for 3 hours. After the reaction was cooled to room temperature, it was diluted with water (20 mL) and ethyl acetate (10 mL), extracted with ethyl acetate (30 mL x 2), the organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product 7-cyclopropyl- 3-methyl-1- (5-oxo-hexane) -1 H - purine -2,6 (3 H, 7 H) - dione was used directly in the next reaction. MS-ESI calculated value [M+H] + 305, measured value 305.

第五步。 the fifth step.

7-環丙基-3-甲基-1-(6，6，6-三氟-5-羥基-5-甲基己基)-1H-嘌呤-2，6(3H，7H)-二酮。 7-Cyclopropyl-3-methyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)-1 H -purine-2,6(3 H ,7 H )- Diketone.

氮氣保護下，將7-環丙基-3-甲基-1-(5-氧代己烷)-1H-嘌呤-2，6(3H，7H)-二酮(200mg，0.660mmol)溶解於無水四氫呋喃(3mL)中，然後依次加入三氟甲基三甲基矽烷(0.2mL，0.990mmol)和氟化銫(20.0mg，0.130mmol)。所得反應液在30℃下反應2小時。然後反應液用水(30mL)稀釋，乙酸乙酯萃取(30mL x 2)，有機相用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用製備TLC板純化(1：2石油醚/乙酸乙酯，Rf=0.3)得到7-環丙基-3-甲基-1-(6，6，6-三氟-5-羥基-5-甲基己基)-1H-嘌呤-2，6(3H，7H)-二酮(150mg，白色固體)，產率：61%。1H NMR：(400MHz，CDCl3)δ7.55(s，1H)，4.13-4.02(m，2H)，3.63-3.61(m，1H)，3.55(s，3H)，2.96(s，1H)，1.90-1.68(m，2H)，1.67-1.64(m，2H)，1.47-1.45(m，2H)，1.28(s，3H)，1.18-1.16(m，2H)，1.06-1.04(m，2H)。MS-ESI計算值[M+H]+ 375，實測值375。 Under the protection of nitrogen, the 7-cyclopropyl-3-methyl-1-(5-oxohexane)-1 H -purine-2,6(3 H ,7 H )-dione (200mg, 0.660mmol ) Was dissolved in anhydrous tetrahydrofuran (3mL), and then trifluoromethyltrimethylsilane (0.2mL, 0.990mmol) and cesium fluoride (20.0mg, 0.130mmol) were added sequentially. The resulting reaction liquid was reacted at 30°C for 2 hours. Then the reaction solution was diluted with water (30mL), extracted with ethyl acetate (30mL x 2), the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and purified with a preparative TLC plate (1:2 petroleum ether/ethyl acetate, Rf=0.3) to obtain 7-cyclopropyl-3-methyl-1-(6,6,6-trifluoro-5-hydroxy-5-methylhexyl)-1 H -purine-2,6(3 H , 7 H )-diketone (150 mg, white solid), yield: 61%. 1 H NMR: (400MHz, CDCl 3 ) δ 7.55 (s, 1H), 4.13-4.02 (m, 2H), 3.63-3.61 (m, 1H), 3.55 (s, 3H), 2.96 (s, 1H) , 1.90-1.68(m, 2H), 1.67-1.64(m, 2H), 1.47-1.45(m, 2H), 1.28(s, 3H), 1.18-1.16(m, 2H), 1.06-1.04(m, 2H). MS-ESI calculated value [M+H] + 375, measured value 375.

實施例48。 Example 48.

7-(環丙基甲基)-1-((4-羥基-4-(三氟甲基)環己基)甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 7-(Cyclopropylmethyl)-1-((4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -purine-2,6(3 H , 7 H )-Diketone.

Figure 105135042-A0305-02-0137-126
Figure 105135042-A0305-02-0137-126
Figure 105135042-A0305-02-0138-128
Figure 105135042-A0305-02-0138-128

第一步。 first step.

1-(1，4-二氧雜螺[4.5]癸烷-8-基甲基)-7-異丙基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1-(1,4-dioxaspiro[4.5]decane-8-ylmethyl)-7-isopropyl-3-methyl-1 H -purine-2,6(3 H , 7 H ) -Diketone.

將1，4-二氧雜螺[4.5]癸烷-8-基甲基甲磺酸酯(250mg，1.00mmol)，7-異丙基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(208mg，1.00mmol)，碘化鉀(15.8mg，0.100mmol)和碳酸鉀(276mg，2.00mmol)溶於無水N，N-二甲基甲醯胺(8mL)中。反應液加熱至120℃，攪拌3小時。反應冷却至20℃，將混合物過濾，濾液減壓濃縮得到粗品1-(1，4-二氧雜螺[4.5]癸烷-8-基甲基)-7-異丙基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(300mg，白色油狀物)，產率：83%。MS-ESI計算值[M+H]+ 362，實測值362。 1,4-Dioxaspiro[4.5]decane-8-yl methyl methanesulfonate (250mg, 1.00mmol), 7-isopropyl-3-methyl-1 H -purine-2,6 (3 H , 7 H )-dione (208mg, 1.00mmol), potassium iodide (15.8mg, 0.100mmol) and potassium carbonate (276mg, 2.00mmol) dissolved in anhydrous N , N -dimethylformamide (8mL) in. The reaction solution was heated to 120°C and stirred for 3 hours. The reaction was cooled to 20°C, the mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain crude 1-(1,4-dioxaspiro[4.5]decane-8-ylmethyl)-7-isopropyl-3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (300 mg of, white oil), yield: 83%. MS-ESI calculated value [M+H] + 362, measured value 362.

第二步。 The second step.

7-異丙基-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮 7-isopropyl-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -purine-2,6(3 H ,7 H )-dione

將1-(1，4-二氧雜螺[4.5]癸烷-8-基甲基)-7-異丙基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(300mg，0.828mmol)溶於丙酮(10mL)中，加入鹽酸(0.5mL)。反應液在室溫攪拌30分鐘。向反應液中加入水，用飽和碳酸氫鈉水溶液(10mL)調至pH值至7，用乙酸乙酯(10mL x 3)萃取，合併有機相，用飽和氯化鈉(20mL x 3)洗滌，無水硫酸鈉乾燥，過 濾，濾液減壓濃縮，用矽膠柱色譜法分離純化(乙酸乙酯，Rf=0.3)，得到7-異丙基-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮(180mg，白色油狀物)，產率：68%。MS-ESI計算值[M+H]+ 319，實測值319。 1- (1,4-dioxaspiro [4.5] decan-8-yl-methyl) -7-isopropyl-3-methyl -1 H - purine -2,6 (3 H, 7 H )-Diketone (300 mg, 0.828 mmol) was dissolved in acetone (10 mL), and hydrochloric acid (0.5 mL) was added. The reaction solution was stirred at room temperature for 30 minutes. Add water to the reaction solution, adjust the pH to 7 with saturated sodium bicarbonate aqueous solution (10 mL), extract with ethyl acetate (10 mL x 3), combine the organic phases, and wash with saturated sodium chloride (20 mL x 3), Dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and separate and purify by silica gel column chromatography (ethyl acetate, Rf=0.3) to obtain 7-isopropyl-3-methyl-1-((4-oxocyclohexyl) ) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (180 mg of, white oil), yield: 68%. MS-ESI calculated value [M+H] + 319, measured value 319.

第三步。 third step.

7-異丙基-3-甲基-1-((4-羥基-4-(三氟甲基)環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮。 7-isopropyl-3-methyl-1-((4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-1 H -purine-2,6(3 H , 7 H )- Diketone.

將7-異丙基-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮(122mg，0.382mmol)和氟化銫(11.5mg，0.0763mmol)溶於無水四氫呋喃(3mL)中，在氮氣保護下加入三氟甲基三甲基矽烷(95.0mg，0.640mmol)。反應液加熱至30℃，攪拌12小時。然後加入鹽酸水溶液(1N，5mL)，再攪拌30分鐘。向反應液中加入水，用飽和碳酸氫鈉水溶液(10mL)調至pH值至7，用乙酸乙酯(10mL x 3)萃取，合併有機相，用飽和氯化鈉(20mL x 3)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用製備高效液相色譜分離純化，得到7-異丙基-3-甲基-1-((4-羥基-4-(三氟甲基)環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮(80.0mg，白色固體)，產率：53%。1H NMR：(400MHz，Methonal-d 4 )δ8.10(s，1H)，5.06-5.00(m，1H)，4.08-3.91(m，2H)，3.55(s，3H)，2.17-2.00(m，2H)，1.88-1.84(m，2H)，1.61-1.40(m，6H)，1.59-1.57(m，5H)。MS-ESI計算值[M+H]+ 389，實測值389。 7-isopropyl-3-methyl-1 - ((4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (122mg, 0.382mmol) And cesium fluoride (11.5mg, 0.0763mmol) were dissolved in anhydrous tetrahydrofuran (3mL), and trifluoromethyltrimethylsilane (95.0mg, 0.640mmol) was added under the protection of nitrogen. The reaction solution was heated to 30°C and stirred for 12 hours. Then an aqueous hydrochloric acid solution (1N, 5 mL) was added and stirred for another 30 minutes. Add water to the reaction solution, adjust the pH to 7 with saturated sodium bicarbonate aqueous solution (10 mL), extract with ethyl acetate (10 mL x 3), combine the organic phases, and wash with saturated sodium chloride (20 mL x 3), Dry with anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and separate and purify by preparative high performance liquid chromatography to obtain 7-isopropyl-3-methyl-1-((4-hydroxy-4-(trifluoromethyl) hexyl-yl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (80.0 mg, white solid), yield: 53%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 8.10 (s, 1H), 5.06-5.00 (m, 1H), 4.08-3.91 (m, 2H), 3.55 (s, 3H), 2.17-2.00 ( m, 2H), 1.88-1.84 (m, 2H), 1.61-1.40 (m, 6H), 1.59-1.57 (m, 5H). MS-ESI calculated value [M+H] + 389, measured value 389.

實施例49。 Example 49.

7-(環丙基甲基)-1-((4-羥基-4-(三氟甲基)環己基)甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 7-(Cyclopropylmethyl)-1-((4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -purine-2,6(3 H , 7 H )-Diketone.

Figure 105135042-A0305-02-0140-130
Figure 105135042-A0305-02-0140-130

第一步。 first step.

1-(1，4-二氧雜螺[4.5]癸烷-8-基甲基)-7-(環丙基甲基)-3-甲基-1H-嘌呤2，6(3H，7H)-二酮。 1-(1,4-Dioxaspiro[4.5]decane-8-ylmethyl)-7-(cyclopropylmethyl)-3-methyl-1 H -purine 2,6(3 H , 7 H )-Diketone.

將(環丙基甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(220mg，1.00mmol)，1，4-二氧雜螺[4.5]癸烷-8-基甲基甲磺酸酯(250mg，1.00mmol)，碘化鉀(15.8mg，0.100mmol)和碳酸鉀(276mg，2.00mmol)溶於無水N，N-二甲基甲醯胺(8mL)中，反應液加熱至120℃，攪拌3小時。反應冷却至20℃，將混合物過濾並減壓濃縮得到粗品1-(1，4-二氧雜螺[4.5]癸烷-8-基甲基)-7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(300mg，白色油狀物)，產率：80%。MS-ESI計算值[M+H]+ 375，實測值375。 The (cyclopropylmethyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (220mg, 1.00mmol), 1,4- dioxaspiro [4.5] Decane-8-yl methyl methanesulfonate (250mg, 1.00mmol), potassium iodide (15.8mg, 0.100mmol) and potassium carbonate (276mg, 2.00mmol) are dissolved in anhydrous N , N -dimethylformamide ( 8mL), the reaction solution was heated to 120°C and stirred for 3 hours. The reaction was cooled to 20°C, the mixture was filtered and concentrated under reduced pressure to obtain crude 1-(1,4-dioxaspiro[4.5]decane-8-ylmethyl)-7-(cyclopropylmethyl)-3 - methyl - -1 H - purine -2,6 (3 H, 7 H) - dione (300 mg of, white oil), yield: 80%. MS-ESI calculated value [M+H] + 375, measured value 375.

第二步。 The second step.

7-(環丙基甲基)-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮。 7- (cyclopropylmethyl) -3-methyl-1 - ((4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione.

將1-(1，4-二氧雜螺[4.5]癸烷-8-基甲基)-3，7-二甲基-1H-嘌呤-2，6(3H，7H)-二酮(300mg，0.802mmol)溶於丙酮(10mL)中，加入 鹽酸(0.5mL)，將混合物置於室溫攪拌30分鐘。向反應液中加入水(30mL)，用飽和碳酸氫鈉水溶液(10mL)調至PH值至7，用乙酸乙酯(10mL x 3)萃取，合併有機相，用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用矽膠柱色譜法分離純化(乙酸乙酯，Rf=0.3)，得到7-(環丙基甲基)-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮(180mg，白色油狀物)，產率：75%。MS-ESI計算值[M+H]+ 331，實測值331。 1- (1,4-dioxaspiro [4.5] decan-8-yl-methyl) -3,7-dimethyl -1 H - purine -2,6 (3 H, 7 H) - two Ketone (300 mg, 0.802 mmol) was dissolved in acetone (10 mL), hydrochloric acid (0.5 mL) was added, and the mixture was stirred at room temperature for 30 minutes. Water (30mL) was added to the reaction solution, adjusted to pH 7 with saturated aqueous sodium bicarbonate (10mL), extracted with ethyl acetate (10mL x 3), combined the organic phases, dried with anhydrous sodium sulfate, filtered, and the filtrate Concentrated under reduced pressure, separated and purified by silica gel column chromatography (ethyl acetate, Rf=0.3) to obtain 7-(cyclopropylmethyl)-3-methyl-1-((4-oxocyclohexyl)methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (180 mg of, white oil), yield: 75%. MS-ESI calculated value [M+H] + 331, measured value 331.

第三步。 third step.

7-(環丙基甲基)-1-((4-羥基-4-(三氟甲基)環己基)甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 7-(Cyclopropylmethyl)-1-((4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -purine-2,6(3 H , 7 H )-Diketone.

將7-(環丙基甲基)-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮(126mg，0.382mmol)和氟化銫(11.5mg，0.0763mmol)溶於無水四氫呋喃(3mL)中，在氮氣保護下加入三氟甲基三甲基矽烷(95.0mg，0.640mmol)。將混合物置於30℃下攪拌12小時。然後加入1N鹽酸水溶液(5mL)，再繼續攪拌30分鐘。向反應液中加入水(30mL)，用飽和碳酸氫鈉水溶液(10mL)調至pH值至7，用乙酸乙酯(10mL x 3)萃取，合併有機相，用飽和氯化鈉(30mL x 2)洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，用製備高效液相色譜分離純化，得到7-(環丙基甲基)-1-((4-羥基-4-(三氟甲基)環己基)甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(80.0mg，白色固體)，產率：53%。1H NMR：(400MHz，Methonal-d 4 )δ 8.16-8.15(m，1H)，4.24-4.22(m，2H)，4.08-3.91(m，2H)，3.57(s，3H)，2.18-2.07(m，2H)，1.85-1.82(m，2H)，1.61-1.47(m，6H)，0.64-0.60(m，2H)，0.50-0.48(m，2H)。MS-ESI計算值[M+H]+ 401，實測值401。 7- (cyclopropylmethyl) -3-methyl-1 - ((4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (126 mg of , 0.382mmol) and cesium fluoride (11.5mg, 0.0763mmol) were dissolved in anhydrous tetrahydrofuran (3mL), and trifluoromethyltrimethylsilane (95.0mg, 0.640mmol) was added under the protection of nitrogen. The mixture was stirred at 30°C for 12 hours. Then 1N aqueous hydrochloric acid solution (5 mL) was added, and stirring was continued for another 30 minutes. Water (30mL) was added to the reaction solution, adjusted to pH 7 with saturated aqueous sodium bicarbonate (10mL), extracted with ethyl acetate (10mL x 3), combined organic phases, and saturated sodium chloride (30mL x 2 ) Was washed, dried with anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and separated and purified by preparative high performance liquid chromatography to obtain 7-(cyclopropylmethyl)-1-((4-hydroxy-4-(trifluoromethyl) ) cyclohexyl) methyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (80.0 mg, white solid), yield: 53%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 8.16-8.15 (m, 1H), 4.24-4.22 (m, 2H), 4.08-3.91 (m, 2H), 3.57 (s, 3H), 2.18-2.07 (m, 2H), 1.85-1.82 (m, 2H), 1.61-1.47 (m, 6H), 0.64-0.60 (m, 2H), 0.50-0.48 (m, 2H). MS-ESI calculated value [M+H] + 401, measured value 401.

實施例50。 Example 50.

Figure 105135042-A0305-02-0142-235
Figure 105135042-A0305-02-0142-235

7-(環丙基甲基)-1-((4-羥基-4-(三氟甲基)環己基)甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(900mg，2.25mmol)，通過製備SFC來分離得到兩個異構體。分離條件：色譜柱：AD 250mm x 30mm，5um流動相：A：超臨界二氧化碳，B：甲醇(0.05%氨水)，A：B=55：45流速：40mL/min波長：220nm。產物1(異構體1，第一個峰)(600mg，白色固體)，產率：100%。1H NMR：(400MHz，DMSO-d 6)δ8.11(s，1H)，5.62(s，1H)，4.08(d，J=7.6Hz，2H)，3.88(d，J=7.6Hz，2H)，3.43(s，3H)，2.05-2.04(m，1H)，1.85-1.82(m，2H)，1.48-1.45(m，2H)，1.33-1.32(m，2H)，1.30-1.28(m，3H)，0.48-0.46(m，2H)，0.41-0.39(m，2H)。MS-ESI計算值[M+H]+401，實測值401。 7-(Cyclopropylmethyl)-1-((4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -purine-2,6(3 H , 7 H )-diketone (900 mg, 2.25 mmol), two isomers were separated by preparing SFC. Separation conditions: Chromatographic column: AD 250mm x 30mm, 5um mobile phase: A: supercritical carbon dioxide, B: methanol (0.05% ammonia), A: B=55:45 Flow rate: 40mL/min Wavelength: 220nm. Product 1 (isomer 1, first peak) (600 mg, white solid), yield: 100%. 1 H NMR: (400MHz, DMSO- d 6 ) δ8.11 (s, 1H), 5.62 (s, 1H), 4.08 (d, J = 7.6 Hz, 2H), 3.88 (d, J = 7.6 Hz, 2H ), 3.43 (s, 3H), 2.05-2.04 (m, 1H), 1.85-1.82 (m, 2H), 1.48-1.45 (m, 2H), 1.33-1.32 (m, 2H), 1.30-1.28 (m , 3H), 0.48-0.46 (m, 2H), 0.41-0.39 (m, 2H). MS-ESI calculated value [M+H] + 401, measured value 401.

產物2(異構體2，第二個峰)(300mg，白色固體)，產率100%。1H NMR：(400MHz，DMSO-d 6)δ8.11(s，1H)，5.62(s，1H)，4.09(d，J=7.6Hz，2H)，3.74(d，J=7.6Hz，2H)，3.42(s，3H)，1.69-1.45(m，3H)，1.45-1.29(m，7H)，0.48-0.46(m，2H)，0.41-0.39(m，2H)。MS-ESI計算值[M+H]+401，實測值401。 Product 2 (isomer 2, second peak) (300 mg, white solid), yield 100%. 1 H NMR: (400MHz, DMSO- d 6 ) δ 8.11 (s, 1H), 5.62 (s, 1H), 4.09 (d, J = 7.6 Hz, 2H), 3.74 (d, J = 7.6 Hz, 2H ), 3.42 (s, 3H), 1.69-1.45 (m, 3H), 1.45-1.29 (m, 7H), 0.48-0.46 (m, 2H), 0.41-0.39 (m, 2H). MS-ESI calculated value [M+H] + 401, measured value 401.

實施例51。 Example 51.

Figure 105135042-A0305-02-0143-132
Figure 105135042-A0305-02-0143-132

第一步。 first step.

1-(1，4-二氧雜螺[4，5]癸烷-8-基甲基-7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-(1,4-Dioxaspiro[4,5]decane-8-ylmethyl-7-(cyclopropylmethyl)-3-methyl-1 H -purine-2,6-( 3 H , 7 H )-dione.

將1，4-二氧雜螺[4，5]癸烷-8-基甲基甲磺酸酯(682mg，2.72mmol)，7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-(3H，7H)-二酮(500mg，2.27mmol)及碘化鉀(37.7mg，0.227mmol)溶於N，N-二甲基甲醯胺(10mL)中，加入碳酸鉀(627mg，4.54mmol)，反應130℃加熱回流4小時。反應液冷却至室溫，過濾，濾液減壓濃縮，得到的粗產品1-(1，4-二氧雜螺[4，5]癸烷-8-基甲基-7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-(3H，7H)-二酮(1.10g，黃色油狀)。MS-ESI計算值[M+H]+ 375，實測值375。 1,4-Dioxaspiro[4,5]decane-8-ylmethanesulfonate (682mg, 2.72mmol), 7-(cyclopropylmethyl)-3-methyl-1 H - purine -2,6- (3 H, 7 H) - dione (500mg, 2.27mmol) and potassium iodide (37.7mg, 0.227mmol) was dissolved in N, N - dimethylformamide (10 mL) was added Potassium carbonate (627mg, 4.54mmol), the reaction was heated to reflux at 130°C for 4 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product 1-(1,4-dioxaspiro[4,5]decane-8-ylmethyl-7-(cyclopropylmethyl) Yl)-3-methyl-1 H -purine-2,6-(3 H , 7 H )-dione (1.10g, yellow oil). MS-ESI calculated value [M+H] + 375, measured The value is 375.

第二步。 The second step.

7-(環丙基甲基)-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮。 7- (cyclopropylmethyl) -3-methyl-1 - ((4-oxo-cyclohexyl) methyl) -1 H - purine -2,6- (3 H, 7 H) - dione.

將1-(1，4-二氧雜螺[4，5]癸烷-8-基甲基-7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-(3H，7H)-二酮(1.20g，2.09mmol)溶於丙酮(12mL)中，加入4N鹽酸水溶液(3mL)。反應室溫攪拌過夜，加入水(20mL)，用乙酸乙酯(30mL x 3)萃取，有機相用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到的產品用矽膠柱色譜法純化(1：1石油醚/乙酸乙酯，Rf=0.3)得到產物7-(環丙基甲基)-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮(52.0mg，黃色固體)，產率：8%。MS-ESI計算值[M+H]+ 331，實測值331。 The 1-(1,4-dioxaspiro[4,5]decane-8-ylmethyl-7-(cyclopropylmethyl)-3-methyl-1 H -purine-2,6- (3 H , 7 H )-diketone (1.20g, 2.09mmol) was dissolved in acetone (12mL), 4N aqueous hydrochloric acid solution (3mL) was added. The reaction was stirred at room temperature overnight, water (20mL) was added, and ethyl acetate ( 30mL x 3) extraction, the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product obtained was purified by silica gel column chromatography (1:1 petroleum ether/ethyl acetate, Rf=0.3) to obtain the product 7-( Cyclopropylmethyl)-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -purine-2,6-(3 H , 7 H )-dione (52.0 mg, yellow Solid), yield: 8%. MS-ESI calculated value [M+H] + 331, measured value 331.

第三步。 third step.

將7-(環丙基甲基)-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6-(3H，7H)-二酮(100mg，0.303mmol)溶於四氫呋喃(5mL)，-78℃時在氮氣保護下緩慢加入甲基格氏試劑(3M乙醚溶液，0.600mL，1.81mmol)，-78℃攪拌半小時，接著0℃反應2小時。加入水(10mL)，用乙酸乙酯(30mL x 3)萃取，有機相用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到粗產品用製備高效液相色譜純化得產物1(26.0mg，白色固體)(異構體1，第一個峰)，產率：25%。1H NMR：(400MHz，Methonal-d 4)δ7.99(s，1H)，4.19(d，J=7.6Hz，2H)，3.90(d，J=7.6Hz，2H)，3.54(s，3H)，1.90-1.79(m，1H)，1.70-1.61(m，4H)，1.45-1.36(m，3H)，1.27-1.16(m，5H)，0.65-0.55(m，2H)，0.49-0.42(m，2H)。MS-ESI計算值[M+H-H2O]+ 329，實測值329。 The 7-(cyclopropylmethyl)-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -purine-2,6-(3 H , 7 H )-dione ( 100mg, 0.303mmol) dissolved in tetrahydrofuran (5mL), slowly add methyl Grignard reagent (3M ether solution, 0.600mL, 1.81mmol) under nitrogen protection at -78℃, stir for half an hour at -78℃, and then react at 0℃ 2 hours. Water (10mL) was added, extracted with ethyl acetate (30mL x 3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. Purified by preparative high performance liquid chromatography to obtain product 1 (26.0mg, white Solid) (Isomer 1, first peak), yield: 25%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.99 (s, 1H), 4.19 (d, J = 7.6 Hz, 2H), 3.90 (d, J = 7.6 Hz, 2H), 3.54 (s, 3H) ), 1.90-1.79(m, 1H), 1.70-1.61(m, 4H), 1.45-1.36(m, 3H), 1.27-1.16(m, 5H), 0.65-0.55(m, 2H), 0.49-0.42 (m, 2H). MS-ESI calculated value [M+HH 2 O] + 329, measured value 329.

產物2(42.0mg，白色固體)(異構體2，第二個峰)，產率：40%。1H NMR：(400MHz，Methonal-d 4)δ7.99(s，1H)，4.19(d，J=7.6Hz，2H)，3.89(d，J=7.6Hz，2H)，3.54(s，3H)，1.81-1.70(m，1H)，1.69-1.62(m，2H)，1.51-1.41(m，4H)，1.39-1.25(m，3H)，1.15(s， 3H)，0.63-0.56(m，2H)，0.48-0.42(m，2H)。MS-ESI計算值[M+H-H2O]+ 329，實測值329。 Product 2 (42.0 mg, white solid) (isomer 2, second peak), yield: 40%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.99 (s, 1H), 4.19 (d, J = 7.6 Hz, 2H), 3.89 (d, J = 7.6 Hz, 2H), 3.54 (s, 3H) ), 1.81-1.70(m, 1H), 1.69-1.62(m, 2H), 1.51-1.41(m, 4H), 1.39-1.25(m, 3H), 1.15(s, 3H), 0.63-0.56(m , 2H), 0.48-0.42 (m, 2H). MS-ESI calculated value [M+HH 2 O] + 329, measured value 329.

實施例52。 Example 52.

1-((4-羥基-1-甲基-4-(三氟甲基)環己基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7-dimethyl-1 H -purine-2,6-(3 H ,7 H )-Diketone.

Figure 105135042-A0305-02-0145-133
Figure 105135042-A0305-02-0145-133

第一步。 first step.

1-((4-羥基-1-甲基-4-(三氟甲基)環己基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7-dimethyl-1 H -purine-2,6-(3 H ,7 H )-Diketone.

(4-羥基-1-(甲氧基甲基)-4-(三氟甲基)環己基)甲基甲磺酸酯(100mg，0.349mmol，7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-(3H，7H)-二酮(76.9mg，0.349mmol)，碘化鉀(5.8mg，0.0349mmol)和碳酸鉀(149mg，1.05mmol)溶於無水N，N-二甲基甲醯胺(5mL)中。反應液微波加熱至150℃，反應2小時。反應液冷却至20℃，過濾，用製備高效液相色譜純化，得到1-((4-羥基-1-甲基-4-(三氟甲基)環己基)甲基)-3，7-二甲基-1H-嘌呤-2，6-(3H，7H)-二酮(10.0mg，白色固體)，產率：6%。1H NMR：(400MHz，DMSO-d 6 )δ8.13(s，1H)，4.12(s，2H)，3.94(s，1H)，3.43-3.38(m，4H)，3.31(s，3H)，3.19(s， 3H)，1.56-1.45(m，8H)，1.43-1.31(m，1H)，0.51-0.49(m，2H)，0.44-0.42(m，2H)。 (4-Hydroxy-1-(methoxymethyl)-4-(trifluoromethyl)cyclohexyl)methanesulfonate (100mg, 0.349mmol, 7-(cyclopropylmethyl)-3- methyl -1 H - purine -2,6- (3 H, 7 H) - dione (76.9mg, 0.349mmol), potassium iodide (5.8mg, 0.0349mmol) and potassium carbonate (149mg, 1.05mmol) was dissolved in dry N , N -dimethylformamide (5mL). The reaction solution was heated to 150°C in microwave and reacted for 2 hours. The reaction solution was cooled to 20°C, filtered, and purified by preparative high performance liquid chromatography to obtain 1-((4 -Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3,7-dimethyl-1 H -purine-2,6-(3 H ,7 H )-dione (10.0mg, white solid), yield: 6%. 1 H NMR: (400MHz, DMSO- d 6 )δ8.13(s, 1H), 4.12(s, 2H), 3.94(s, 1H), 3.43 -3.38(m, 4H), 3.31(s, 3H), 3.19(s, 3H), 1.56-1.45(m, 8H), 1.43-1.31(m, 1H), 0.51-0.49(m, 2H), 0.44 -0.42 (m, 2H).

MS-ESI計算值[M+H]+ 445，實測值445。。 MS-ESI calculated value [M+H] + 445, measured value 445. .

實施例53。 Example 53.

7-(環丙基甲基)-1-((4-羥基-1-甲基-4-(三氟甲基)環己基)甲基)-3-甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 7-(Cyclopropylmethyl)-1-((4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -purine-2, 6-(3 H , 7 H )-dione.

Figure 105135042-A0305-02-0146-135
Figure 105135042-A0305-02-0146-135

第一步。 first step.

7-(環丙基甲基)-1-((4-羥基-1-甲基-4-(三氟甲基)環己基)甲基)-3-甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 7-(Cyclopropylmethyl)-1-((4-hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methyl)-3-methyl-1 H -purine-2, 6-(3 H , 7 H )-dione.

(4-羥基-1-甲基-4-(三氟甲基)環己基)甲基甲磺酸酯(100mg，0.344mmol)，7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-(3H，7H)-二酮(75.9mg，0.344mmol)，碘化鉀(5.70mg，0.0344mmol)和碳酸鉀(47.6mg，0.344mmol)溶於無水N，N-二甲基甲醯胺(5mL)中。反應液加熱至150℃，微波反應4小時。反應液冷却至20℃，過濾，濃縮，然後用製備高效液相色譜純化，得到7-(環丙基甲基)-1-((4-羥基-1-甲基-4-(三氟甲基)環己基)甲基)-3-甲基-1H-嘌呤-2，6-(3H，7H)-二酮(10.0mg，白色固體)，產率：7%。1H NMR：(400MHz，DMSO-d 6 )δ8.13(s，1H)，4.13-4.09(m，2H)，3.83(s，1H)，3.43(s，3H)，3.34(s，2H)， 1.67-1.53(m，6H)，1.23-1.20(m，3H)，0.88(s，3H)，0.50-0.42(m，4H)。MS-ESI計算值[M+H]+ 415，實測值415。 (4-Hydroxy-1-methyl-4-(trifluoromethyl)cyclohexyl)methanesulfonate (100mg, 0.344mmol), 7-(cyclopropylmethyl)-3-methyl-1 H - purine -2,6- (3 H, 7 H) - dione (75.9mg, 0.344mmol), potassium iodide (5.70mg, 0.0344mmol) and potassium carbonate (47.6mg, 0.344mmol) was dissolved in anhydrous N, N -Dimethylformamide (5 mL). The reaction solution was heated to 150° C. and reacted in a microwave for 4 hours. The reaction solution was cooled to 20°C, filtered, concentrated, and then purified by preparative high performance liquid chromatography to obtain 7-(cyclopropylmethyl)-1-((4-hydroxy-1-methyl-4-(trifluoromethyl) yl) cyclohexyl) methyl) -3-methyl -1 H - purine -2,6- (3 H, 7 H) - dione (10.0 mg, white solid), yield: 7%. 1 H NMR: (400MHz, DMSO- d 6 )δ8.13(s, 1H), 4.13-4.09(m, 2H), 3.83(s, 1H), 3.43(s, 3H), 3.34(s, 2H) , 1.67-1.53 (m, 6H), 1.23-1.20 (m, 3H), 0.88 (s, 3H), 0.50-0.42 (m, 4H). MS-ESI calculated value [M+H] + 415, measured value 415.

實施例54。 Example 54.

7-(環丙基甲基)-3-甲基-1-((5-甲基-2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-4-基)甲基)-1H-嘌呤2，6-(3H，7H)-二酮。 7-(Cyclopropylmethyl)-3-methyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl )Methyl)-1H-purine 2,6-(3H,7H)-dione.

Figure 105135042-A0305-02-0147-136
Figure 105135042-A0305-02-0147-136

第一步。 first step.

1-(4-(溴甲基)-5-甲基噻唑-2-基)乙酮。 1-(4-(Bromomethyl)-5-methylthiazol-2-yl)ethanone.

將1-(4，5-二甲基吡啶-2-基)乙酮(200mg，1.29mmol)，N-溴代丁二醯亞胺(229mg，1.29mmol)，偶氮二異丁腈(21.2mg，0.129mmol)溶於四氯化碳(20mL)中，80℃反應12小時。加入飽和硫代硫酸鈉溶液(30mL)淬滅反應。用二氯甲烷萃取(10mL x 3)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到1-(4-(溴甲基)-5-甲基噻唑-2-基)乙酮(200mg，黃色油狀)，產率：46%。MS-ESI計算值[M+H]+ 234，236，實測值234，236。 Combine 1-(4,5-dimethylpyridin-2-yl)ethanone (200mg, 1.29mmol), N -bromosuccinimide (229mg, 1.29mmol), azobisisobutyronitrile (21.2 mg, 0.129mmol) was dissolved in carbon tetrachloride (20mL) and reacted at 80°C for 12 hours. The reaction was quenched by adding saturated sodium thiosulfate solution (30 mL). It was extracted with dichloromethane (10mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 1-(4-(bromomethyl)-5-methylthiazol-2-yl)ethanone (200mg, Yellow oil), yield: 46%. MS-ESI calculated value [M+H] + 234,236, measured value 234,236.

第二步。 The second step.

1-((2-乙醯基-5-甲基噻唑-4-基)甲基)-7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-(3H，7H)-二酮。 1-((2-Acetyl-5-methylthiazol-4-yl)methyl)-7-(cyclopropylmethyl)-3-methyl-1 H -purine-2,6-(3H , 7H)-Diketone.

將1-(4-(溴甲基)-5-甲基噻唑-2-基)乙酮(200mg，0.598mmol)，7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-二酮(132mg，0.598mmol)，碘化鉀(19.8mg，0.119mmol)和碳酸鉀(248mg，1.79mmol)溶於N，N-二甲基甲醯胺(10mL)中。反應液升溫至120℃，攪拌3小時。冷却至室溫，過濾，濾液減壓濃縮用製備TLC板分離純化(1：1石油醚/乙酸乙酯，Rf值=0.4)得到1-((2-乙醯基-5-甲基噻唑-4-基)甲基)-7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-(3H，7H)-二酮(100mg，黃色固體)，產率：45%。1H NMR：(400MHz，Methonal-d 4 )δ8.02(s，1H)，5.35(s，2H)，4.21(d，J=7.6Hz，2H)，3.56(s，3H)，2.66(s，3H)，2.60(s，3H)，1.46-1.41(m，1H)，0.65-0.61(m，2H)，0.60-0.48(m，2H)。MS-ESI計算值[M+H]+ 374，實測值374。 Add 1-(4-(bromomethyl)-5-methylthiazol-2-yl)ethanone (200mg, 0.598mmol), 7-(cyclopropylmethyl)-3-methyl-1 H -purine -2,6-dione (132 mg, 0.598 mmol), potassium iodide (19.8 mg, 0.119 mmol) and potassium carbonate (248 mg, 1.79 mmol) were dissolved in N , N -dimethylformamide (10 mL). The reaction solution was heated to 120°C and stirred for 3 hours. Cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure and separated and purified by preparative TLC plate (1:1 petroleum ether/ethyl acetate, Rf value=0.4) to obtain 1-((2-acetyl-5-methylthiazole- 4-yl)methyl)-7-(cyclopropylmethyl)-3-methyl- 1H -purine-2,6-( 3H , 7H )-dione (100mg, yellow solid), yield Rate: 45%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 8.02 (s, 1H), 5.35 (s, 2H), 4.21 (d, J = 7.6 Hz, 2H), 3.56 (s, 3H), 2.66 (s , 3H), 2.60 (s, 3H), 1.46-1.41 (m, 1H), 0.65-0.61 (m, 2H), 0.60-0.48 (m, 2H). MS-ESI calculated value [M+H] + 374, measured value 374.

第三步。 third step.

7-(環丙基甲基)-3-甲基-1-((5-甲基-2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-4-基)甲基)-1H-嘌呤2，6-(3H，7H)-二酮。 7-(Cyclopropylmethyl)-3-methyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl ) methyl) -1 H - purin-2,6- (3 H, 7 H) - dione.

將1-((2-乙醯基-5-甲基噻唑-4-基)甲基)-7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-(3H，7H)-二酮(100mg，0.267mmol)，氟化銫(40.6mg，0.267mmol)溶於四氫呋喃(10mL)中，室溫下加入三甲基-三氟甲基-矽烷(114mg，0.803mmol)，攪拌12小時。加入水(20mL)淬滅反應。用乙酸乙酯萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用製備型高效液相色譜純化，得到7-(環丙基甲基)-3-甲基-1-((5-甲基-2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-4-基)甲基)-1H-嘌呤2，6-(3H，7H)-二酮(50.0mg，黃色固體)， 產率：42%。1H NMR：(400MHz，Methonal-d 4 )δ8.11(s，1H)，5.33(s，2H)，4.23(d，J=7.6Hz，2H)，3.57(s，3H)，2.64(s，3H)，1.81(s，3H)，1.45-1.41(m，1H)，0.65-0.61(m，2H)，0.60-0.49(m，2H)。MS-ESI計算值[M+H]+ 444，實測值444。 1-((2-Acetyl-5-methylthiazol-4-yl)methyl)-7-(cyclopropylmethyl)-3-methyl-1 H -purine-2,6-( 3 H , 7 H )-dione (100mg, 0.267mmol), cesium fluoride (40.6mg, 0.267mmol) dissolved in tetrahydrofuran (10mL), add trimethyl-trifluoromethyl-silane (114mg , 0.803mmol) and stirred for 12 hours. The reaction was quenched by adding water (20 mL). Extract with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purified by preparative high performance liquid chromatography to obtain 7-(cyclopropylmethyl)-3-methyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropane) 2-yl) thiazol-4-yl) methyl) -1 H - purin-2,6- (3 H, 7 H) - dione (50.0 mg, yellow solid), yield: 42%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 8.11 (s, 1H), 5.33 (s, 2H), 4.23 (d, J = 7.6 Hz, 2H), 3.57 (s, 3H), 2.64 (s , 3H), 1.81 (s, 3H), 1.45-1.41 (m, 1H), 0.65-0.61 (m, 2H), 0.60-0.49 (m, 2H). MS-ESI calculated value [M+H] + 444, measured value 444.

實施例55。 Example 55.

7-(環丙基甲基)-3-甲基-1-((6-(1，1，1-三氟-2-羥基丙烷-2-基)吡啶-3-基)甲基)-1H-嘌呤2，6-(3H，7H)-二酮。 7-(Cyclopropylmethyl)-3-methyl-1-((6-(1,1,1-trifluoro-2-hydroxypropan-2-yl)pyridin-3-yl)methyl)- 1 H -purine 2,6-(3 H ,7 H )-dione.

Figure 105135042-A0305-02-0149-137
Figure 105135042-A0305-02-0149-137

第一步。 first step.

1-[5-(溴甲基)-2-吡啶基]乙酮。 1-[5-(Bromomethyl)-2-pyridyl]ethanone.

將1-(5-甲基-2-吡啶基)乙酮(500mg，3.70mmol)，N-溴代丁二醯亞胺(658mg，3.70mmol)，偶氮二異丁腈(182mg，1.11mmol)溶於四氯化碳(20mL)中，90℃反應12小時。加入飽和硫代硫酸鈉溶液(30mL)淬滅反應。用二氯甲烷萃取(10mL x 3)，無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到1-[5-(溴甲基)-2-吡啶基]乙酮(125mg，黃色油狀)，產率：16%。MS-ESI計算值[M+H]+ 214和216，實測值214和216。 Combine 1-(5-methyl-2-pyridyl)ethanone (500mg, 3.70mmol), N -bromosuccinimide (658mg, 3.70mmol), azobisisobutyronitrile (182mg, 1.11mmol) ) Was dissolved in carbon tetrachloride (20mL) and reacted at 90°C for 12 hours. The reaction was quenched by adding saturated sodium thiosulfate solution (30 mL). Extract with dichloromethane (10mL x 3), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain 1-[5-(bromomethyl)-2-pyridyl]ethanone (125mg, yellow oil). Yield: 16%. MS-ESI calculated values [M+H] + 214 and 216, and measured values 214 and 216.

第二步。 The second step.

1-[(6-乙醯基-3-吡啶基)甲基]-7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-二酮。 1-[(6-Acetyl-3-pyridyl)methyl]-7-(cyclopropylmethyl)-3-methyl-1 H -purine-2,6-dione.

將1-[5-(溴甲基)-2-吡啶基]乙酮(100mg，0.467mmol)，7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-二酮(103mg，0.467mmol)，碘化鉀(15.5mg，0.0934mmol)和碳酸鉀(193mg，1.40mmol)溶於N，N-二甲基甲醯胺(10mL)中。反應液升溫至120℃，攪拌3小時。冷却至室溫，過濾，濾液減壓濃縮用製備TLC板分離純化(乙酸乙酯，Rf值=0.4)得到1-[(6-乙醯基-3-吡啶基)甲基]-7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-二酮(50.0mg，黃色固體)，產率：30%。MS-ESI計算值[M+H]+ 354，實測值354。 The 1-[5-(bromomethyl)-2-pyridyl]ethanone (100mg, 0.467mmol), 7-(cyclopropylmethyl)-3-methyl- 1H -purine-2,6- The diketone (103 mg, 0.467 mmol), potassium iodide (15.5 mg, 0.0934 mmol) and potassium carbonate (193 mg, 1.40 mmol) were dissolved in N , N -dimethylformamide (10 mL). The reaction solution was heated to 120°C and stirred for 3 hours. Cool to room temperature, filter, and concentrate the filtrate under reduced pressure. Separate and purify with a preparative TLC plate (ethyl acetate, Rf value = 0.4) to obtain 1-[(6-acetyl-3-pyridyl)methyl]-7-( Cyclopropylmethyl)-3-methyl-1 H -purine-2,6-dione (50.0 mg, yellow solid), yield: 30%. MS-ESI calculated value [M+H] + 354, measured value 354.

第三步。 third step.

7-(環丙基甲基)-3-甲基-1-((6-(1，1，1-三氟-2-羥基丙烷-2-基)吡啶-3-基)甲基)-1H-嘌呤2，6-(3H，7H)-二酮。 7-(Cyclopropylmethyl)-3-methyl-1-((6-(1,1,1-trifluoro-2-hydroxypropan-2-yl)pyridin-3-yl)methyl)- 1 H -purine 2,6-(3 H ,7 H )-dione.

將1-[(6-乙醯基-3-吡啶基)甲基]-7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-二酮(100mg，0.283mmol)，氟化銫(43.0mg，0.283mmol)溶於四氫呋喃(10mL)中，室溫下加入三甲基-三氟甲基-矽烷(60.4mg，0.424mmol)，攪拌12小時。加入水(20mL)淬滅反應。用乙酸乙酯萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用製備型高效液相色譜純化，得到7-(環丙基甲基)-3-甲基-1-((6-(1，1，1-三氟-2-羥基丙烷-2-基)吡啶-3-基)甲基)-1H-嘌呤2，6-(3H，7H)-二酮(40.0mg，黃色固體)，產率：32%。1H NMR：(400MHz，Methonal-d 4 )δ9.00(s，1H)，8.80-8.72(m，1H)，8.46(s，1H)，8.35-8.29(m，1H)，5.43(s，2H)，4.28(d，J=7.6Hz，2H)，3.58(s，3H)，1.95(s，3H)，1.50-1.46(m，1H)，0.68-0.64(m，2H)，0.53-0.51(m，2H)。MS-ESI計算值[M+H]+ 424，實測值424。 1-[(6-Acetyl-3-pyridyl)methyl]-7-(cyclopropylmethyl)-3-methyl- 1H -purine-2,6-dione (100mg, 0.283 mmol), cesium fluoride (43.0 mg, 0.283 mmol) was dissolved in tetrahydrofuran (10 mL), trimethyl-trifluoromethyl-silane (60.4 mg, 0.424 mmol) was added at room temperature, and the mixture was stirred for 12 hours. The reaction was quenched by adding water (20 mL). Extract with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purified by preparative high performance liquid chromatography to obtain 7-(cyclopropylmethyl)-3-methyl-1-((6-(1,1,1-trifluoro-2-hydroxypropan-2-yl) pyridin-3-yl) methyl) -1 H - purin-2,6- (3 H, 7 H) - dione (40.0 mg, yellow solid), yield: 32%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 9.00 (s, 1H), 8.80-8.72 (m, 1H), 8.46 (s, 1H), 8.35 to 8.29 (m, 1H), 5.43 (s, 2H), 4.28(d, J =7.6Hz, 2H), 3.58(s, 3H), 1.95(s, 3H), 1.50-1.46(m, 1H), 0.68-0.64(m, 2H), 0.53-0.51 (m, 2H). MS-ESI calculated value [M+H] + 424, measured value 424.

實施例56。 Example 56.

7-(環丙基甲基)-3-甲基-1-((5-甲基-2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-4-基)甲基)-1H-嘌呤2，6-(3H，7H)-二酮。 7-(Cyclopropylmethyl)-3-methyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl ) methyl) -1 H - purin-2,6- (3 H, 7 H) - dione.

Figure 105135042-A0305-02-0151-139
Figure 105135042-A0305-02-0151-139

第一步。 first step.

1-[(5-乙醯基-2-吡啶基)甲基]-7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-二酮。 1-[(5-Acetyl-2-pyridyl)methyl]-7-(cyclopropylmethyl)-3-methyl-1 H -purine-2,6-dione.

將1-[6-(溴甲基)-3-吡啶基]乙酮(100mg，0.467mmol)，7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-二酮(103mg，0.467mmol)，碘化鉀(15.5mg，0.0934mmol)和碳酸鉀(193mg，1.40mmol)溶於N，N-二甲基甲醯胺(10mL)中。反應液升溫至120℃，攪拌3小時。冷却至室溫，過濾，濾液減壓濃縮用製備TLC板分離純化(乙酸乙酯，Rf值=0.5)得到1-[(5-乙醯基-2-吡啶基)甲基]-7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-二酮(50.0mg，黃色固體)，產率：30%。MS-ESI計算值[M+H]+ 354，實測值354。 The 1-[6-(bromomethyl)-3-pyridyl]ethanone (100mg, 0.467mmol), 7-(cyclopropylmethyl)-3-methyl- 1H -purine-2,6- The diketone (103 mg, 0.467 mmol), potassium iodide (15.5 mg, 0.0934 mmol) and potassium carbonate (193 mg, 1.40 mmol) were dissolved in N , N -dimethylformamide (10 mL). The reaction solution was heated to 120°C and stirred for 3 hours. Cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. It was separated and purified by preparative TLC plate (ethyl acetate, Rf value=0.5) to obtain 1-[(5-acetyl-2-pyridyl)methyl]-7-( Cyclopropylmethyl)-3-methyl-1 H -purine-2,6-dione (50.0 mg, yellow solid), yield: 30%. MS-ESI calculated value [M+H] + 354, measured value 354.

第二步。 The second step.

7-(環丙基甲基)-3-甲基-1-((5-甲基-2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-4-基)甲基)-1H-嘌呤2，6-(3H，7H)-二酮。 7-(Cyclopropylmethyl)-3-methyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropan-2-yl)thiazol-4-yl ) methyl) -1 H - purin-2,6- (3 H, 7 H) - dione.

將1-[(5-乙醯基-2-吡啶基)甲基]-7-(環丙基甲基)-3-甲基-1H-嘌呤-2，6-二酮(100mg，0.283mmol)，氟化銫(43.0mg，0.283mmol)溶於四氫呋喃(10mL)中，室溫下加入三甲基-三氟甲基-矽烷(60.4mg，0.424mmol)，攪拌12小時。加入水(20mL)淬滅反應。用乙酸乙酯萃取(10mL x 3)。合併有機相，用飽和食鹽水洗滌，無水硫酸鈉乾燥，過濾，濾液減壓濃縮。用製備型高效液相色譜純化，得到7-(環丙基甲基)-3-甲基-1-((5-甲基-2-(1，1，1-三氟-2-羥基丙烷-2-基)噻唑-4-基)甲基)-1H-嘌呤2，6-(3H，7H)-二酮(10.0mg，黃色固體)，產率：8%。1H NMR：(400MHz，Methonal-d 4 )δ8.92(s，1H)，8.75(d，J=7.6Hz，1H)，8.09(d，J=7.6Hz，1H)，7.97(s，1H)，5.54(s，2H)，4.21(d，J=7.6Hz，2H)，3.58(s，3H)，1.85(s，3H)，1.45-1.42(m，1H)，0.64-0.59(m，2H)，0.50-0.46(m，2H)。MS-ESI計算值[M+H]+ 424，實測值424。 1-[(5-Acetyl-2-pyridyl)methyl]-7-(cyclopropylmethyl)-3-methyl- 1H -purine-2,6-dione (100mg, 0.283 mmol), cesium fluoride (43.0 mg, 0.283 mmol) was dissolved in tetrahydrofuran (10 mL), trimethyl-trifluoromethyl-silane (60.4 mg, 0.424 mmol) was added at room temperature, and the mixture was stirred for 12 hours. The reaction was quenched by adding water (20 mL). Extract with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Purified by preparative high performance liquid chromatography to obtain 7-(cyclopropylmethyl)-3-methyl-1-((5-methyl-2-(1,1,1-trifluoro-2-hydroxypropane) 2-yl) thiazol-4-yl) methyl) -1 H - purin-2,6- (3 H, 7 H) - dione (10.0 mg, yellow solid), yield: 8%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 8.92 (s, 1H), 8.75 (d, J = 7.6 Hz, 1H), 8.09 (d, J = 7.6 Hz, 1H), 7.97 (s, 1H) ), 5.54 (s, 2H), 4.21 (d, J = 7.6 Hz, 2H), 3.58 (s, 3H), 1.85 (s, 3H), 1.45-1.42 (m, 1H), 0.64-0.59 (m, 2H), 0.50-0.46 (m, 2H). MS-ESI calculated value [M+H] + 424, measured value 424.

實施例57。 Example 57.

1-((4-羥基-4-(三氟甲基)環己基)甲基)-7-異丁基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1-((4-Hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-7-isobutyl-3-methyl-1 H -purine-2,6(3 H ,7 H )- Diketone.

Figure 105135042-A0305-02-0152-141
Figure 105135042-A0305-02-0152-141

第一步。 first step.

1-(1，4-二氧雜螺[4，5]癸烷-8-基甲基)-7-異丁基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1-(1,4-Dioxaspiro[4,5]decane-8-ylmethyl)-7-isobutyl-3-methyl-1 H -purine-2,6(3 H ,7 H )-Diketone.

將1，4-二氧雜螺[4，5]癸烷-8-基甲基甲磺酸酯(1.07g，4.81mmol)，7-異丁基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(1.00g，4.01mmol)及碳酸鉀(647mg，4.81mmol)溶於N，N-二甲基甲醯胺(14mL)中，加入碘化鉀(66.5mg，0.401mmol)，反應130℃加熱回流3小時。反應液直接過濾，濾液減壓濃縮，得到粗產品1-(1，4-二氧雜螺[4，5]癸烷-8-基甲基)-7-異丁基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(粗產品2.56g，棕色油狀)。MS-ESI計算值[M+H]+ 377，實測值377。 1,4-Dioxaspiro[4,5]decane-8-yl methyl methanesulfonate (1.07g, 4.81mmol), 7-isobutyl-3-methyl-1 H -purine- 2,6 (3 H, 7 H) - dione (1.00g, 4.01mmol) and potassium carbonate (647mg, 4.81mmol) was dissolved in N, N - dimethylformamide (14mL) was added potassium iodide (66.5 mg, 0.401mmol), the reaction was heated to reflux at 130°C for 3 hours. The reaction solution was directly filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product 1-(1,4-dioxaspiro[4,5]decane-8-ylmethyl)-7-isobutyl-3-methyl- 1 H - purine -2,6 (3 H, 7 H) - dione (crude product 2.56g, brown oil). MS-ESI calculated value [M+H] + 377, measured value 377.

第二步。 The second step.

7-異丁基-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮。 7-isobutyl-3-methyl-1 - ((4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione.

將1-(1，4-二氧雜螺[4，5]癸烷-8-基甲基)-7-異丁基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(2.50g，6.00mmol)溶於丙酮(12mL)中，加入4N鹽酸水溶液(2mL)。反應30℃攪拌過夜，加入飽和碳酸氫鈉水溶液(8mL)調節pH至7。向反應液中加入水(100mL)，用乙酸乙酯(150mL x 3)萃取，有機相用無水硫酸鈉乾燥，過濾，濾液減壓濃縮，得到的產品用矽膠柱色譜法純化(1：2石油醚/乙酸乙酯，Rf=0.3)得到產物7-異丁基-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮(150mg，白色固體)，產率：13%。1H NMR：(400MHz，Methonal-d 4)δ7.94(s，1H)，4.14(d，J=7.6Hz，2H)，3.99(d，J=7.6Hz，2H)，3.55(s，3H)，2.29-2.38(m，5H),2.20-2.13(m，1H)，2.03-1.98(m，2H),1.53-1.47(m，2H)，0.92(d，J=6.4Hz，6H)。MS-ESI計算值[M+H]+ 333，實測值333。 The 1-(1,4-dioxaspiro[4,5]decane-8-ylmethyl)-7-isobutyl-3-methyl-1 H -purine-2,6(3 H , 7 H )-dione (2.50 g, 6.00 mmol) was dissolved in acetone (12 mL), and 4N aqueous hydrochloric acid solution (2 mL) was added. The reaction was stirred at 30°C overnight, and saturated aqueous sodium bicarbonate solution (8 mL) was added to adjust the pH to 7. Water (100mL) was added to the reaction solution, extracted with ethyl acetate (150mL x 3), the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The product obtained was purified by silica gel column chromatography (1:2 petroleum Ether/ethyl acetate, Rf=0.3) to obtain the product 7-isobutyl-3-methyl-1-((4-oxocyclohexyl)methyl)-1 H -purine-2,6(3 H , 7 H )-diketone (150 mg, white solid), yield: 13%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.94 (s, 1H), 4.14 (d, J = 7.6 Hz, 2H), 3.99 (d, J = 7.6 Hz, 2H), 3.55 (s, 3H) ), 2.29-2.38 (m, 5H), 2.20-2.13 (m, 1H), 2.03-1.98 (m, 2H), 1.53-1.47 (m, 2H), 0.92 (d, J = 6.4 Hz, 6H). MS-ESI calculated value [M+H] + 333, measured value 333.

第三步。 third step.

1-((4-羥基-4-(三氟甲基)環己基)甲基)-7-異丁基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1-((4-Hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-7-isobutyl-3-methyl-1 H -purine-2,6(3 H ,7 H )- Diketone.

將7-異丁基-3-甲基-1-((4-氧環己基)甲基)-1H-嘌呤-2，6(3H，7H)-二酮(150mg，0.450mmol)及氟化銫(8.0mg，0.0450mmol)溶於四氫呋喃(3mL)中，在氮氣保護下緩慢加入三氟甲基三甲基矽烷(950mg，0.640mmol)。反應30℃下攪拌16小時，加入4N鹽酸水溶液(3mL)並25℃攪拌半小時後，加入飽和碳酸氫鈉水溶液(15mL)調節pH至7，加入水(50mL)並用乙酸乙酯(50mL x 3)，有機相用無水硫酸鈉乾燥，減壓濃縮，得到粗產品用製備高效液相色譜純化得產物1-((4-羥基-4-(三氟甲基)環己基)甲基)-7-異丁基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(86.0mg，白色固體)，產率：48%。1H NMR：(400MHz，Methanol-d 4)δ7.93(s，1H)，4.15-4.04(m，2H)，3.89(d，J=7.6Hz，1H)，3.54(s，3H)，2.20-1.98(m，3H)，1.86-1.79(m，2H)，1.61-1.42(m，5H)，0.92(d，J=6.4Hz，6H)。MS-ESI計算值[M+H]+ 403，實測值403。 7-isobutyl-3-methyl-1 - ((4-oxo-cyclohexyl) methyl) -1 H - purine -2,6 (3 H, 7 H) - dione (150mg, 0.450mmol) And cesium fluoride (8.0mg, 0.0450mmol) was dissolved in tetrahydrofuran (3mL), and trifluoromethyltrimethylsilane (950mg, 0.640mmol) was slowly added under the protection of nitrogen. The reaction was stirred at 30°C for 16 hours, 4N aqueous hydrochloric acid (3mL) was added and stirred at 25°C for half an hour, saturated aqueous sodium bicarbonate (15mL) was added to adjust the pH to 7, water (50mL) was added and ethyl acetate (50mL x 3 ), the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. Purified by preparative high performance liquid chromatography to obtain the product 1-((4-hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-7 - isobutyl-3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (86.0 mg, white solid), yield: 48%. 1 H NMR: (400MHz, Methanol- d 4 )δ7.93(s, 1H), 4.15-4.04(m, 2H), 3.89(d, J =7.6Hz, 1H), 3.54(s, 3H), 2.20 -1.98 (m, 3H), 1.86-1.79 (m, 2H), 1.61-1.42 (m, 5H), 0.92 (d, J = 6.4 Hz, 6H). MS-ESI calculated value [M+H] + 403, measured value 403.

實施例58。 Example 58.

Figure 105135042-A0305-02-0154-142
Figure 105135042-A0305-02-0154-142

1-((4-羥基-4-(三氟甲基)環己基)甲基)-7-異丁基-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(900mg，2.24mmol)，通過製備SFC來分離得到兩個異構體。異構體1分離條件：色譜柱：AD 250mm x 30mm，5um流動相：A：超臨界二氧化碳，B：乙醇(0.05%氨水)，A：B=80：20流速：50mL/min波長：220nm。異構體2分離條件：色譜柱：WEEK-1 300mm x 25mm，5um流動相：A：超臨界二氧化碳，B：乙醇(0.05%氨水)，A：B=60：40，流速：60mL/min，波長：220nm。 1-((4-Hydroxy-4-(trifluoromethyl)cyclohexyl)methyl)-7-isobutyl-3-methyl-1 H -purine-2,6(3 H ,7 H )- Dione (900mg, 2.24mmol), two isomers were separated by preparing SFC. Isomer 1 separation conditions: Column: AD 250mm x 30mm, 5um mobile phase: A: supercritical carbon dioxide, B: ethanol (0.05% ammonia), A: B=80:20 Flow rate: 50mL/min Wavelength: 220nm. Isomer 2 separation conditions: Column: WEEK-1 300mm x 25mm, 5um Mobile phase: A: supercritical carbon dioxide, B: ethanol (0.05% ammonia), A: B=60:40, flow rate: 60mL/min, Wavelength: 220nm.

產物1(異構體1，第一個峰)(216mg，白色固體)，產率：36%。1H NMR：(400MHz，DMSO-d 6)δ8.09(s，1H)，5.65(s，1H)，4.07(d，J=7.2Hz，2H)，3.90(d，J=7.2Hz，2H)，3.43(s，3H)，2.14-2.00(m，2H)，1.92-1.80(m，2H)，1.77-1.66(m，2H)，1.52-1.44(m，2H)，1.37-1.30(m，2H)，0.84(d，J=6.4Hz，6H)。MS-ESI計算值[M+H]+ 403，實測值403。 Product 1 (isomer 1, first peak) (216 mg, white solid), yield: 36%. 1 H NMR: (400MHz, DMSO- d 6 ) δ 8.09 (s, 1H), 5.65 (s, 1H), 4.07 (d, J = 7.2 Hz, 2H), 3.90 (d, J = 7.2 Hz, 2H ), 3.43 (s, 3H), 2.14-2.00 (m, 2H), 1.92-1.80 (m, 2H), 1.77-1.66 (m, 2H), 1.52-1.44 (m, 2H), 1.37-1.30 (m , 2H), 0.84 (d, J = 6.4 Hz, 6H). MS-ESI calculated value [M+H] + 403, measured value 403.

產物2(異構體2，第二個峰)(101mg，白色固體)，產率37%。1H NMR：(400MHz，DMSO-d 6)δ8.07(s，1H)，5.64(s，1H)，4.05(d，J=7.6Hz，2H)，3.75(d，J=7.6Hz，2H)，3.42(s，3H)，2.16-2.03(m，1H)，1.71-1.66(m，3H)，1.48-1.30(m，6H)，0.83(d，J=6.4Hz，6H)。MS-ESI計算值[M+H]+ 403，實測值403。 Product 2 (isomer 2, second peak) (101 mg, white solid), yield 37%. 1 H NMR: (400MHz, DMSO- d 6 ) δ 8.07 (s, 1H), 5.64 (s, 1H), 4.05 (d, J = 7.6 Hz, 2H), 3.75 (d, J = 7.6 Hz, 2H ), 3.42 (s, 3H), 2.16-2.03 (m, 1H), 1.71-1.66 (m, 3H), 1.48-1.30 (m, 6H), 0.83 (d, J = 6.4 Hz, 6H). MS-ESI calculated value [M+H] + 403, measured value 403.

實施例59。 Example 59.

7-(2，3-二羥基丙基)-1-(5-乙基-5-羥基庚基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 7-(2,3-Dihydroxypropyl)-1-(5-ethyl-5-hydroxyheptyl)-3-methyl-1 H -purine-2,6(3 H ,7 H )-di ketone.

Figure 105135042-A0305-02-0155-144
Figure 105135042-A0305-02-0155-144
Figure 105135042-A0305-02-0156-146
Figure 105135042-A0305-02-0156-146

第一步。 first step.

7-((2，2-二甲基-1，3-二氧戊環-4-基)甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 7-((2,2-Dimethyl-1,3-dioxolane-4-yl)methyl)-3-methyl-1 H -purine-2,6(3 H ,7 H )- Diketone.

將3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(200mg，1.20mmol)，碳酸鈉(128mg，1.20mmol)，4-(氯甲基)-2，2-二甲基-1，3-二氧戊環(217mg，1.44mmol)和碘化鉀(20.0mg，0.120mmol)溶解於N，N-二甲基甲醯胺(10mL)中。反應液加熱到110℃，反應36小時。加水(30mL)淬滅反應，用乙酸乙酯萃取(10mL x 3)，有機相用飽和食鹽水洗滌(5mL)，無水硫酸鈉乾燥，減壓濃縮，用製備TLC板分離純化(乙酸乙酯，Rf=0.5)得到7-((2，2-二甲基-1，3-二氧戊環-4-基)甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(169mg，黃色固體)，產率：50%。MS-ESI計算值[M+H]+ 281，實測值281。 3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (200mg, 1.20mmol), sodium carbonate (128mg, 1.20mmol), 4- (chloromethyl) -2, 2-Dimethyl-1,3-dioxolane (217 mg, 1.44 mmol) and potassium iodide (20.0 mg, 0.120 mmol) were dissolved in N , N -dimethylformamide (10 mL). The reaction solution was heated to 110°C and reacted for 36 hours. The reaction was quenched by adding water (30 mL), extracted with ethyl acetate (10 mL x 3), the organic phase was washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated and purified with a preparative TLC plate (ethyl acetate, Rf=0.5) to obtain 7-((2,2-dimethyl-1,3-dioxolane-4-yl)methyl)-3-methyl-1 H -purine-2,6(3 H , 7 H )-diketone (169 mg, yellow solid), yield: 50%. MS-ESI calculated value [M+H] + 281, measured value 281.

第二步。 The second step.

乙基5-(7-((2，2-二甲基-1，3-二氧戊環-4-基)甲基)-3-甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤烷-1-基)戊酸乙酯。 Ethyl 5-(7-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)-3-methyl-2,6-dioxo-2,3 , 6,7-Tetrahydro- 1H -purin-1-yl)ethyl valerate.

將7-((2，2-二甲基-1，3-二氧戊環-4-基)甲基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(169mg，0.604mmol)，溴戊酸乙酯(178mg，0.906mmol)，碳酸鉀(167mg，1.21mmol)和碘化鉀(20.0mg，0.0600mmol)溶解於N，N-二甲基甲醯胺(10mL)中。反應液加熱到130℃，反應3小時，過濾，濃縮，用製備TLC板分離純化(乙酸乙酯，Rf=0.4)得到乙基5-(7-((2，2-二甲基-1，3-二氧戊環-4-基)甲基)-3-甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤烷-1-基)戊酸乙酯(124mg，黃色固體)，產率：50%。MS-ESI計算值[M+H]+ 409，實測值409。 7 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) -Diketone (169mg, 0.604mmol), ethyl bromovalerate (178mg, 0.906mmol), potassium carbonate (167mg, 1.21mmol) and potassium iodide (20.0mg, 0.0600mmol) dissolved in N , N -dimethylformamide Amine (10 mL). The reaction solution was heated to 130°C, reacted for 3 hours, filtered, concentrated, and separated and purified with a preparative TLC plate (ethyl acetate, Rf=0.4) to obtain ethyl 5-(7-((2,2-dimethyl-1, 3-Dioxolane-4-yl)methyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -purin-1-yl)pentan Ethyl acid (124 mg, yellow solid), yield: 50%. MS-ESI calculated value [M+H] + 409, measured value 409.

第三步。 third step.

7-((2，2-二甲基-1，3-二氧戊環-4-基)甲基)-1-(5-乙基-5-羥基庚基)-3-甲基-1H-嘌呤-2，6(3H，7H)二酮。 7-((2,2-Dimethyl-1,3-dioxolane-4-yl)methyl)-1-(5-ethyl-5-hydroxyheptyl)-3-methyl-1 H - purine -2,6 (3 H, 7 H) -dione.

將乙基5-(7-((2，2-二甲基-1，3-二氧戊環-4-基)甲基)-3-甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤烷-1-基)戊酸乙酯(30.0mg，0.0750mmol)溶解於無水四氫呋喃(1mL)中，於-65℃條件下慢慢滴入乙基溴化鎂(3M四氫呋喃溶液，0.15mL，0.450mmol)。反應在-65℃反應0.5小時，然後在0℃反應0.5小時。反應液倒入水(5mL)中淬滅，用乙酸乙酯萃取(5mL x 3)，用無水硫酸鈉乾燥，過濾，減壓濃縮，用製備TLC板分離純化(乙酸乙酯，Rf=0.5)得到7-((2，2-二甲基-1，3-二氧戊環-4-基)甲基)-1-(5-乙基-5-羥基庚基)-3-甲基-1H-嘌呤-2，6(3H，7H)二酮(20.0mg，黃色油狀物)，產率：63%。MS-ESI計算值[M+H]+ 423，實測值423。 The ethyl 5-(7-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)-3-methyl-2,6-dioxo-2, 3,6,7-Tetrahydro- 1H -purin-1-yl)ethyl valerate (30.0mg, 0.0750mmol) was dissolved in anhydrous tetrahydrofuran (1mL), and ethyl acetate was slowly added dropwise at -65℃. Base magnesium bromide (3M tetrahydrofuran solution, 0.15 mL, 0.450 mmol). The reaction was carried out at -65°C for 0.5 hours, and then at 0°C for 0.5 hours. The reaction solution was poured into water (5mL) and quenched, extracted with ethyl acetate (5mL x 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified with a preparative TLC plate (ethyl acetate, Rf=0.5) 7-((2,2-Dimethyl-1,3-dioxolane-4-yl)methyl)-1-(5-ethyl-5-hydroxyheptyl)-3-methyl- 1 H - purine -2,6 (3 H, 7 H) -dione (20.0 mg of, yellow oil). yield: 63%. MS-ESI calculated value [M+H] + 423, measured value 423.

第四步。 the fourth step.

7-(2，3-二羥基丙基)-1-(5-乙基-5-羥基庚基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 7-(2,3-Dihydroxypropyl)-1-(5-ethyl-5-hydroxyheptyl)-3-methyl-1 H -purine-2,6(3 H ,7 H )-di ketone.

將7-((2，2-二甲基-1，3-二氧戊環-4-基)甲基)-1-(5-乙基-5-羥基庚基)-3-甲基-1H-嘌呤-2，6(3H，7H)二酮(20.0mg，0.0470mmol)溶解於無水四氫呋喃(1mL)和稀鹽酸(0.3mL)中，在25℃條件下反應36小時。反應完全後，減壓濃縮，用製備TLC板分離純化(8：1乙酸乙酯/甲醇，Rf=0.3)純化得到7-(2，3-二羥基丙基)-1-(5-乙基-5-羥基庚基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(5.0mg，白色固體)，產率：28%。1H NMR：(400MHz，Methonal-d 4)δ7.90(s，1H)，4.57-4.53(m，1H)，4.26-4.22(m，1H)，4.03-3.96(m，3H)，3.58-3.55(m，2H)，3.54(s，3H)，1.66-1.61(m，2H)，1.48-1.44(m，6H)，1.34-1.29(m，2H)，0.85(t，J=8.0Hz，6H)。MS-ESI計算值[M+H]+ 383，實測值383。 7-((2,2-Dimethyl-1,3-dioxolane-4-yl)methyl)-1-(5-ethyl-5-hydroxyheptyl)-3-methyl- 1 H - purine -2,6 (3 H, 7 H) -dione (20.0mg, 0.0470mmol) was dissolved in anhydrous tetrahydrofuran (1 mL) and dilute hydrochloric acid (0.3 mL), the reaction at 25 ℃ 36 hours. After the completion of the reaction, it was concentrated under reduced pressure, separated and purified with a preparative TLC plate (8:1 ethyl acetate/methanol, Rf=0.3) to obtain 7-(2,3-dihydroxypropyl)-1-(5-ethyl) -5-hydroxy-hept-yl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (5.0mg, white solid), yield: 28%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.90 (s, 1H), 4.57-4.53 (m, 1H), 4.26-4.22 (m, 1H), 4.03-3.96 (m, 3H), 3.58- 3.55 (m, 2H), 3.54 (s, 3H), 1.66-1.61 (m, 2H), 1.48-1.44 (m, 6H), 1.34-1.29 (m, 2H), 0.85 (t, J = 8.0Hz, 6H). MS-ESI calculated value [M+H] + 383, measured value 383.

實施例60。 Example 60.

1-(5-乙基-5-羥基庚基)-7-(2-羥基乙基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1- (5-ethyl-5-hydroxy-heptyl) -7- (2-hydroxyethyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione.

Figure 105135042-A0305-02-0158-148
Figure 105135042-A0305-02-0158-148

第一步。 first step.

7-(2-羥基乙基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 7- (2-hydroxyethyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione.

將3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(1.00g，6.00mmol)，碳酸鉀(830mg，6.00mmol)溶解於N，N-二甲基甲醯胺(10mL)中。反應液加熱到80℃反應0.5小時，加入2-溴乙醇(900mg，7.20mmol)。反應液加熱至130℃反應過夜。反應液濃縮得到粗品7-(2-羥基乙基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮直接用於下一步。 3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (1.00g, 6.00mmol), potassium carbonate (830mg, 6.00mmol) was dissolved in N, N - dimethylformamide Amide (10 mL). The reaction solution was heated to 80°C for 0.5 hour, and 2-bromoethanol (900mg, 7.20mmol) was added. The reaction solution was heated to 130°C and reacted overnight. The reaction mixture was concentrated to give crude 7- (2-hydroxyethyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione was used directly in the next step.

第二步。 The second step.

乙基5-(7-(2-羥基乙基)-3-甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)戊酸乙酯。 Ethyl 5-(7-(2-hydroxyethyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -purin-1-yl)pentanoic acid Ethyl ester.

7-(2-羥基乙基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(1.05g，5.00mmol)，5-溴戊酸乙酯(1.25g，6.00mmol)和碳酸鉀(1.66g，12.0mmol)溶解於N，N-二甲基甲醯胺(3mL)中。反應液加熱到130℃反應3小時。把反應液倒入水(20mL)中淬滅反應，用乙酸乙酯萃取(20mL x 3)。合併有機相，用無水硫酸鈉乾燥，過濾，減壓濃縮，用製備TLC板分離純化得到乙基5-(7-(2-羥基乙基)-3-甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)戊酸乙酯(700mg，黃色油狀物)，產率：35%。MS-ESI計算值[M+H]+ 339，實測值339。 7-(2-Hydroxyethyl)-3-methyl-1 H -purine-2,6(3 H , 7 H )-dione (1.05g, 5.00mmol), ethyl 5-bromovalerate (1.25 g, 6.00 mmol) and potassium carbonate (1.66 g, 12.0 mmol) were dissolved in N , N -dimethylformamide (3 mL). The reaction solution was heated to 130°C for 3 hours. The reaction solution was poured into water (20 mL) to quench the reaction, and extracted with ethyl acetate (20 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified with a preparative TLC plate to obtain ethyl 5-(7-(2-hydroxyethyl)-3-methyl-2,6-dioxo Ethyl -2,3,6,7-tetrahydro- 1H -purin-1-yl)valerate (700 mg, yellow oil), yield: 35%. MS-ESI calculated value [M+H] + 339, measured value 339.

第三步。 third step.

1-(5-乙基-5-羥基庚基)-7-(2-羥基乙基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1- (5-ethyl-5-hydroxy-heptyl) -7- (2-hydroxyethyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione.

將乙基5-(7-(2-羥基乙基)-3-甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤-1-基)戊酸乙酯(700mg，2.07mmol)溶解於無水四氫呋喃(7mL)中，於-78℃條件下慢慢滴加乙基溴化鎂(3M四氫呋喃溶液，7mL，2.10mmol)。反應液在-78℃反應1小時。反應液倒入水(20mL)中淬滅，用乙酸乙酯萃取(30mL x 3)。有機相用無水硫酸鈉乾燥，過濾，減壓濃 縮，用高效液相色譜法分離純化得到1-(5-乙基-5-羥基庚基)-7-(2-羥基乙基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(110mg，白色固體)，產率：15%。1H NMR：(400MHz，Methonal-d 4 )：δ7.90(s，1H)，4.41(t，J=5.0Hz，2H)，4.00(t，J=7.6Hz，2H)，3.87(t，J=5.0Hz，2H)，3.54(s，3H)，1.68-1.59(m，2H)，1.50-1.42(m，5H)，1.39-1.29(m，3H)，0.95-0.77(m，6H)。MS-ESI計算值[M+H-H2O]+ 335，實測值335。 Ethyl 5-(7-(2-hydroxyethyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl)pentan Ethyl acid (700mg, 2.07mmol) was dissolved in anhydrous tetrahydrofuran (7mL), and ethylmagnesium bromide (3M tetrahydrofuran solution, 7mL, 2.10mmol) was slowly added dropwise at -78°C. The reaction solution was reacted at -78°C for 1 hour. The reaction solution was poured into water (20 mL) and quenched, and extracted with ethyl acetate (30 mL x 3). The organic phase was dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by high performance liquid chromatography to obtain 1-(5-ethyl-5-hydroxyheptyl)-7-(2-hydroxyethyl)-3- methyl -1 H - purine -2,6 (3 H, 7 H) - dione (110 mg, white solid), yield: 15%. 1 H NMR: (400MHz, Methonal- d 4 ): δ 7.90 (s, 1H), 4.41 (t, J = 5.0 Hz, 2H), 4.00 (t, J = 7.6 Hz, 2H), 3.87 (t, J = 5.0Hz, 2H), 3.54 (s, 3H), 1.68-1.59 (m, 2H), 1.50-1.42 (m, 5H), 1.39-1.29 (m, 3H), 0.95-0.77 (m, 6H) . MS-ESI calculated value [M+HH 2 O] + 335, measured value 335.

實施例61。 Example 61.

1-(5-乙基-5-羥基庚醇)-7-(2-羥基-3-((2-羥基乙基)(甲基)氨基)丙基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1-(5-Ethyl-5-hydroxyheptanol)-7-(2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)-3-methyl-1 H- purine -2,6 (3 H, 7 H) - dione.

Figure 105135042-A0305-02-0160-150
Figure 105135042-A0305-02-0160-150

第一步。 first step.

1-溴-3-((2-羥基乙基)(甲基)氨基)丙-2-醇。 1-Bromo-3-((2-hydroxyethyl)(methyl)amino)propan-2-ol.

將2-(甲基氨基)乙醇(135mg，1.80mmol)溶解於N，N-二甲基甲醯胺(5mL)中，加入2-(溴甲基)環氧乙烷(206mg，1.51mmol)室溫下反應1.5小時。反應完全後，該反應液直接用於下一步反應。 Dissolve 2-(methylamino)ethanol (135mg, 1.80mmol) in N , N -dimethylformamide (5mL), and add 2-(bromomethyl)oxirane (206mg, 1.51mmol) React at room temperature for 1.5 hours. After the reaction is complete, the reaction solution is directly used for the next reaction.

第二步。 The second step.

7-(2-羥基-3-((2-羥基乙基)(甲基)氨基)丙基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 7-(2-Hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)-3-methyl-1 H -purine-2,6(3 H ,7 H )-dione .

向1-溴-3-((2-羥基乙基)(甲基)氨基)丙-2-醇的溶液中加入3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(100mg，0.602mmol)，碳酸鈉(64.0mg，0.602mmol)和碘化鉀(10.0mg，0.0600mmol)。反應液加熱到80℃，反應10小時。反應完全後，過濾，減壓濃縮，得到粗品7-(2-羥基-3-((2-羥基乙基)(甲基)氨基)丙基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮直接用於下一步。MS-ESI計算值[M+H]+ 298，實測值298。 A solution of 1- bromo-3 - ((2-hydroxyethyl) (methyl) amino) propan-2-ol was added 3-methyl -1 H - purine -2,6 (3 H, 7 H) -Diketone (100 mg, 0.602 mmol), sodium carbonate (64.0 mg, 0.602 mmol) and potassium iodide (10.0 mg, 0.0600 mmol). The reaction solution was heated to 80°C and reacted for 10 hours. After the reaction is complete, filter and concentrate under reduced pressure to obtain crude 7-(2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)-3-methyl-1 H -purine-2 , 6( 3H , 7H )-dione was used directly in the next step. MS-ESI calculated value [M+H] + 298, measured value 298.

第三步。 third step.

乙基5-(7-(2-羥基-3-((2-羥基乙基)(甲基)氨基)丙基)-3-甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤烷-1-基)戊酸乙酯。 Ethyl 5-(7-(2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)-3-methyl-2,6-dioxo-2,3,6 , 7-Tetrahydro- 1H -purin-1-yl)ethyl valerate.

將7-(2-羥基-3-((2-羥基乙基)(甲基)氨基)丙基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(170mg，0.572mmol)，溴戊酸乙酯(169mg，0.858mmol)，碳酸鉀(158mg，1.14mmol)和碘化鉀(10.0mg，0.0570mmol)溶解於N，N-二甲基甲醯胺(5mL)中。反應液加熱到130℃，反應3小時。把反應液倒入水(5mL)中淬滅，用乙酸乙酯萃取(10mL x 3)。合併有機相，用無水硫酸鈉乾燥，過濾，濃縮，用製備TLC板分離純化(8：1二氯甲烷/甲醇，Rf=0.4)得乙基5-(7-(2-羥基-3-((2-羥基乙基)(甲 基)氨基)丙基)-3-甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤烷-1-基)戊酸乙酯(118mg，黃色油狀物)，產率：49%。MS-ESI計算值[M+H]+ 426，實測值426。 7- (2-hydroxy-3 - ((2-hydroxyethyl) (methyl) amino) propyl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - two Ketone (170mg, 0.572mmol), ethyl bromovalerate (169mg, 0.858mmol), potassium carbonate (158mg, 1.14mmol) and potassium iodide (10.0mg, 0.0570mmol) were dissolved in N , N -dimethylformamide ( 5mL). The reaction solution was heated to 130°C and reacted for 3 hours. The reaction solution was poured into water (5 mL) for quenching, and extracted with ethyl acetate (10 mL x 3). The organic phases were combined, dried with anhydrous sodium sulfate, filtered, concentrated, and separated and purified with a preparative TLC plate (8:1 dichloromethane/methanol, Rf=0.4) to obtain ethyl 5-(7-(2-hydroxy-3-() (2-Hydroxyethyl)(methyl)amino)propyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro- 1H -purin-1-yl ) Ethyl valerate (118 mg, yellow oil), yield: 49%. MS-ESI calculated value [M+H] + 426, measured value 426.

第四步。 the fourth step.

1-(5-乙基-5-羥基庚醇)-7-(2-羥基-3-((2-羥基乙基)(甲基)氨基)丙基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮。 1-(5-Ethyl-5-hydroxyheptanol)-7-(2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)-3-methyl-1 H- purine -2,6 (3 H, 7 H) - dione.

將乙基5-(7-(2-羥基-3-((2-羥基乙基)(甲基)氨基)丙基)-3-甲基-2，6-二氧代-2，3，6，7-四氫-1H-嘌呤烷-1-基)戊酸乙酯(100mg，0.235mmol)溶解於無水四氫呋喃(4mL)中，在-65℃條件下慢慢滴入乙基溴化鎂(3M四氫呋喃溶液，0.47mL，1.41mmol)。反應在-65℃反應0.5時，然後在0℃反應0.5小時。反應完全後，反應液倒入水(5mL)中淬滅，用乙酸乙酯萃取(5mL x 3)，用無水硫酸鈉乾燥，過濾，減壓濃縮，用製備TLC板分離純化(6：1乙酸乙酯/甲醇，Rf=0.3)純化得1-(5-乙基-5-羥基庚醇)-7-(2-羥基-3-((2-羥基乙基)(甲基)氨基)丙基)-3-甲基-1H-嘌呤-2，6(3H，7H)-二酮(3.0mg，白色固體)，產率：3%。1H NMR：(400MHz，Methonal-d 4 )δ7.92(s，1H)，4.91-4.59(m，2H)，4.52-4.39(m，3H)，4.30-4.25(m，2H)，4.03-3.99(m，2H)，3.88-3.84(m，2H)，3.55(s，3H)，2.90(s，3H)，1.68-1.59(m，4H)，1.48-1.45(m，6H)，0.88-0.84(m，6H)。MS-ESI計算值[M+H]+ 440，實測值440。 The ethyl 5-(7-(2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)-3-methyl-2,6-dioxo-2,3, 6,7-Tetrahydro- 1H -purin-1-yl)ethyl valerate (100mg, 0.235mmol) was dissolved in anhydrous tetrahydrofuran (4mL), and ethyl bromide was slowly added dropwise at -65℃ Magnesium (3M tetrahydrofuran solution, 0.47 mL, 1.41 mmol). The reaction was carried out at -65°C for 0.5 hours, and then at 0°C for 0.5 hours. After the reaction was complete, the reaction solution was poured into water (5mL) for quenching, extracted with ethyl acetate (5mL x 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified with a preparative TLC plate (6:1 acetic acid) Ethyl/methanol, Rf=0.3) purified to obtain 1-(5-ethyl-5-hydroxyheptanol)-7-(2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl yl) -3-methyl -1 H - purine -2,6 (3 H, 7 H) - dione (3.0mg, white solid), yield: 3%. 1 H NMR: (400MHz, Methonal- d 4 ) δ 7.92 (s, 1H), 4.91-4.59 (m, 2H), 4.52-4.39 (m, 3H), 4.30-4.25 (m, 2H), 4.03- 3.99 (m, 2H), 3.88-3.84 (m, 2H), 3.55 (s, 3H), 2.90 (s, 3H), 1.68-1.59 (m, 4H), 1.48-1.45 (m, 6H), 0.88- 0.84 (m, 6H). MS-ESI calculated value [M+H] + 440, measured value 440.

實驗例1：體外評價PDE2磷酸二酯酶抑制活性。 Experimental Example 1: Evaluation of PDE2 phosphodiesterase inhibitory activity in vitro.

實驗目的：通過熒光偏振分析法檢測AMP/GMP抗體上取代的AlexaFluor 633熒光染料來檢測反應體系中產生的AMP/GMP濃度，計算待測化合物的PDE2磷酸二酯酶抑制IC50值。 The purpose of the experiment: to detect the AlexaFluor 633 fluorescent dye substituted on the AMP/GMP antibody by fluorescence polarization analysis to detect the AMP/GMP concentration produced in the reaction system, and to calculate the PDE2 phosphodiesterase inhibition IC 50 value of the test compound.

實驗材料。 Experimental Materials.

測定緩沖溶液：10mM三羥甲基氨基甲烷鹽酸緩衝液，pH 7.5，5mM氯化鎂，0.01%聚氧乙烯月桂醚，1mM二硫蘇糖醇和1% DMSO。酶：使用N端GST標簽用杆狀病毒在昆蟲Sf9細胞中表達重組全長人PDE2A蛋白。底物：1μM cGMP。 Assay buffer solution: 10 mM tris hydrochloric acid buffer, pH 7.5, 5 mM magnesium chloride, 0.01% polyoxyethylene lauryl ether, 1 mM dithiothreitol and 1% DMSO. Enzyme: Use N-terminal GST tag to express recombinant full-length human PDE2A protein in insect Sf9 cells with baculovirus. Substrate: 1μM cGMP.

檢測方法。 Detection method.

Transcreener® AMP2/GMP2抗體，AMP2/GMP2 AlexaFluor 633熒光染料。 Transcreener® AMP 2 /GMP 2 antibody, AMP2/GMP2 AlexaFluor 633 fluorescent dye.

實驗操作。 Experimental operation.

將新鮮製備的緩衝溶液配置酶溶液，然後加入到反應孔穴中，通過Echo550非接觸式納升級聲波移液系統加入待測化合物的DMSO溶液，然後室溫下預溫育10分鐘，加入底物(1μM cGMP)引發反應，室溫反應一小時。然後加入檢測系統(Transcreener® AMP2/GMP2抗體，AMP2/GMP2 AlexaFluor 633熒光染料)，室溫下反應90分鐘，然後使用Ex/Em 620/688檢測熒光偏振。 Prepare the freshly prepared buffer solution to configure the enzyme solution, and then add it to the reaction cavity, add the DMSO solution of the test compound through the Echo550 non-contact nano-upgraded sonic pipetting system, and then pre-incubate at room temperature for 10 minutes, and add the substrate ( 1μM cGMP) to initiate the reaction and react at room temperature for one hour. Then add the detection system (Transcreener® AMP 2 /GMP 2 antibody, AMP2/GMP2 AlexaFluor 633 fluorescent dye), react at room temperature for 90 minutes, and then use Ex/Em 620/688 to detect the fluorescence polarization.

熒光偏振強度通過AMP/GMP標準曲綫換算成nM濃度，然後計算相對DMSO空白的相對酶活性抑制，利用Prism軟件包(GraphPad Software,San Diego California,USA)計算IC50值和曲綫。 Fluorescence polarization intensity was converted to nM concentration by the AMP/GMP standard curve, and then the relative enzyme activity inhibition relative to the DMSO blank was calculated, and the IC50 value and curve were calculated using Prism software package (GraphPad Software, San Diego California, USA).

實驗結果：見表1。 Experimental results: see Table 1.

Figure 105135042-A0305-02-0163-151
Figure 105135042-A0305-02-0163-151
Figure 105135042-A0305-02-0164-153
Figure 105135042-A0305-02-0164-153
Figure 105135042-A0305-02-0165-154
Figure 105135042-A0305-02-0165-154
Figure 105135042-A0305-02-0166-159
Figure 105135042-A0305-02-0166-159

結論：本發明化合物具有顯著甚至意料不到的PDE2A蛋白酶抑制活性。 Conclusion: The compound of the present invention has significant or even unexpected PDE2A protease inhibitory activity.

實驗例2：體外評價化合物對LPS誘導大鼠血液中TNF-α的影響。 Experimental Example 2: In vitro evaluation of the compound's effect on LPS-induced TNF-α in the blood of rats.

實驗目的：在體外檢測化合物對LPS誘導大鼠血液中TNF-α的影響，評估化合物對大鼠血液中LPS誘導TNF-α的抑制作用。 Experimental purpose: to detect the effect of the compound on LPS-induced TNF-α in rat blood in vitro, and to evaluate the inhibitory effect of the compound on LPS-induced TNF-α in rat blood.

實驗材料：Sprague Dawley大鼠(雄性，210~260g，8~10周齡，上海斯萊克)；Rat TNF-alpha Quantikine ELISA Kit(R&D,#SRTA00)。 Experimental materials: Sprague Dawley rats (male, 210~260g, 8~10 weeks old, Shanghai Slack); Rat TNF-alpha Quantikine ELISA Kit (R&D, #SRTA00).

實驗操作：配製濃度為1mM的待測化合物溶液，分別在48孔細胞培養板中加入40μL(化合物終濃度為100uM)。大鼠用異氟烷麻醉後，於心 臟採血(肝素抗凝)。將血液加入已經加好待測化合物的48孔板中，每孔320μL。將48孔板放置於細胞培養箱中，孵育30分鐘後取出，加入40μL LPS溶液(100ug/mL)，混勻後放置於培養箱中繼續孵育。5小時後取出48孔板，血樣轉移至1.5mL離心管中，置於離心機中離心(4,500rpm,4℃,5分鐘)，分離上層得血漿，分裝後速凍，保存在-80度冰箱。第二天按照試劑盒說明書操作用R&D ELISA試劑盒進行血漿樣品中TNF-α水平檢測。 Experimental operation: prepare a solution of the test compound at a concentration of 1 mM, and add 40 μL to a 48-well cell culture plate (the final concentration of the compound is 100 uM). After the rats were anesthetized with isoflurane, Yu Xin Visceral blood collection (heparin anticoagulation). Add blood to a 48-well plate to which the compound to be tested has been added, 320 μL per well. Place the 48-well plate in the cell incubator, incubate for 30 minutes, take it out, add 40μL of LPS solution (100ug/mL), mix well and place in the incubator to continue incubating. After 5 hours, take out the 48-well plate, transfer the blood sample to a 1.5mL centrifuge tube, place it in a centrifuge (4,500rpm, 4°C, 5 minutes), separate the upper layer to obtain the plasma, aliquot and quickly freeze, and store in a -80 degree refrigerator . On the second day, the R&D ELISA kit was used to detect the level of TNF-α in plasma samples according to the kit instructions.

實驗結果：見表2。 Experimental results: see Table 2.

Figure 105135042-A0305-02-0167-158
Figure 105135042-A0305-02-0167-158

結論：本發明化合物具有顯著甚至意料不到的TNF-α抑制活性。 Conclusion: The compound of the present invention has significant or unexpected TNF-α inhibitory activity.

實驗例3：化合物藥代動力學評價。 Experimental example 3: Evaluation of compound pharmacokinetics.

實驗目的：測試化合物在SD大鼠體內藥代動力學。 Experimental purpose: To test the pharmacokinetics of the compound in SD rats.

實驗材料：Sprague Dawley大鼠(雄性，200-300g,7~9周齡，上海斯萊克)。 Experimental materials: Sprague Dawley rats (male, 200-300g, 7-9 weeks old, Shanghai Slack).

實驗操作：以標準方案測試化合物靜脉注射及口服給藥後的嚙齒類動物藥代特徵，實驗中候選化合物配成澄清溶液，給予大鼠單次靜脉注射及口服給藥。靜注及口服溶媒為一定比例的羥丙基β環糊精水溶液或生理鹽水溶液。收集24小時內的全血樣品，3000g離心15分鐘，分離上清得血漿樣品，加入4倍體積含內標的乙腈溶液沉澱蛋白，離心取上清液加入等倍體積的水再離心取上清進樣，以LC-MS/MS分析方法定量分析血藥濃度，並計算藥代參數，如達峰濃度，達峰時間，清除率，半衰期，藥時曲綫下面積，生物利用度等。 Experimental operation: The rodent pharmacokinetic characteristics of the compound after intravenous injection and oral administration were tested by a standard protocol. In the experiment, the candidate compound was prepared into a clear solution and given to rats by a single intravenous injection and oral administration. The vehicle for intravenous injection and oral administration is a certain proportion of hydroxypropyl β cyclodextrin aqueous solution or physiological saline solution. Collect whole blood samples within 24 hours, centrifuge at 3000g for 15 minutes, separate the supernatant to obtain a plasma sample, add 4 times the volume of acetonitrile solution containing internal standard to precipitate the protein, centrifuge to take the supernatant, add the same volume of water, and centrifuge to take the supernatant into Sample, quantitative analysis of blood drug concentration by LC-MS/MS analysis method, and calculation of pharmacokinetic parameters, such as peak concentration, peak time, clearance rate, half-life, area under the drug-time curve, bioavailability, etc.

實驗結果：見表3。 Experimental results: see Table 3.

Figure 105135042-A0305-02-0168-160
Figure 105135042-A0305-02-0168-160
Figure 105135042-A0305-02-0169-161
Figure 105135042-A0305-02-0169-161

結論：本發明化合物可以顯著提高大鼠藥代動力學單項或部分指標。 Conclusion: The compound of the present invention can significantly improve single or partial indexes of rat pharmacokinetics.

實驗例4：MCD小鼠模型體內藥理學研究。 Experimental Example 4: In vivo pharmacological study of MCD mouse model.

主要任務：評估化合物1對非酒精性脂肪性肝炎模型MCD小鼠肝臟組織學，TG水平，TNF-α、mRNA水平和炎症因子(IL-1β水平；血漿ALT/AST水平，炎症因子(TNF-α，IL-1β)水平的影響。 Main task: To evaluate the effect of compound 1 on the liver histology, TG level, TNF-α, mRNA level and inflammatory factor (IL-1β level; plasma ALT/AST level, inflammatory factor (TNF- α, IL-1β) level.

擬解决的問題：測試化合物1對MCD小鼠肝臟組織學，TG水平，TNF-αmRNA水平和炎症因子(IL-1β)水平；血漿ALT/AST水平，炎症因子(TNF-α，IL-1β)水平的影響，評估化合物對非酒精性脂肪性肝炎模型小鼠的肝功能保護，降低肝臟炎症的作用。 Problem to be solved: Test compound 1 on liver histology, TG level, TNF-αmRNA level and inflammatory factor (IL-1β) level of MCD mice; plasma ALT/AST level, inflammatory factor (TNF-α, IL-1β) To evaluate the effect of the compound on the liver function protection of non-alcoholic steatohepatitis model mice and reduce liver inflammation.

實驗模型：小鼠蛋氨酸和膽鹼缺失飲食誘導的非酒精性脂肪性肝炎模型(Methionine-choline-deficient diet treated mice，MCD mice) Experimental model: Non-alcoholic steatohepatitis model induced by methionine and choline deficient diet in mice (Methionine-choline-deficient diet treated mice, MCD mice)

實驗動物：C57BL/6J小鼠(雄性，4周齡購入，上海斯萊克) Experimental animals: C57BL/6J mice (male, purchased at 4 weeks old, Shanghai Slack)

實驗試劑： Experimental reagents:

1.MCD飼料(Research Diets,# A02082002B) 1.MCD feed (Research Diets, # A02082002B)

2.MCD對照飼料(Research Diets,# A02082003B) 2.MCD control feed (Research Diets, # A02082003B)

3.RNeasy® Mini RNA提取試劑盒(Qiagen,#74104) 3.RNeasy® Mini RNA extraction kit (Qiagen, #74104)

4.High Capacity cDNA逆轉錄試劑盒(ABI,# 4368814) 4. High Capacity cDNA Reverse Transcription Kit (ABI, # 4368814)

5.TaqMan® Gene Expression Assay TNF-α(ABI,Assay ID：Mm00443258_m1) 5. TaqMan® Gene Expression Assay TNF-α (ABI, Assay ID: Mm00443258_m1)

6.TaqMan® Endogenous Control 18S(ABI,Assay ID：Mm03928990_g1) 6. TaqMan® Endogenous Control 18S (ABI, Assay ID: Mm03928990_g1)

7.TaqMan® Fast Universal PCR Mater Mix(ABI,# 4366072) 7.TaqMan® Fast Universal PCR Mater Mix(ABI,# 4366072)

8.One cOmplete,EDTA-free protease inhibitor tablet(Roche,# 11873580001) 8.One cOmplete,EDTA-free protease inhibitor tablet(Roche,# 11873580001)

9.BCA蛋白定量試劑盒(Pierce,# 23227) 9. BCA protein quantification kit (Pierce, # 23227)

10.MSD炎症因子檢測試劑盒(Meso Scale Discovery,#K15A0H-2) 10. MSD inflammatory factor detection kit (Meso Scale Discovery, #K15A0H-2)

11.Triglyceride Quantification Kit(BioVision,#K622-100) 11.Triglyceride Quantification Kit(BioVision,#K622-100)

12.Hematoxylin(Baso,# BA4097) 12.Hematoxylin(Baso,# BA4097)

13.Eosin(Baso,# BA4099) 13.Eosin(Baso,# BA4099)

實驗方法。 experimental method.

1.實驗流程。 1. Experimental process.

Figure 105135042-A0305-02-0170-163
Figure 105135042-A0305-02-0170-163

2.體內實驗方法。 2. In vivo experimental methods.

購買4周齡C57BL/6J小鼠，適應環境4周後開始特殊飼料飼養。特殊飼料飼養1周後開始口服給藥的預適應。預適應方法：按照5mL/kg口服溶媒對照0.5%(w/v)羥丙基甲基纖維素。預適應一周後隨機分組，進行口服給藥，分組與給藥信息如下：MCD對照飲食+溶媒對照(p.o.tid,2wk) n=8 Purchase 4 weeks old C57BL/6J mice and start feeding them with special feed after 4 weeks of acclimatization. Pre-adaptation for oral administration started after feeding on special feed for 1 week. Pre-adaptation method: According to 5mL/kg oral vehicle control 0.5% (w/v) hydroxypropyl methylcellulose. One week after pre-adaptation, they were randomly grouped for oral administration. The grouping and administration information is as follows: MCD control diet + vehicle control (p.o.tid, 2wk) n=8

MCD飲食+溶媒對照(p.p.tid,2wk) n=8 MCD diet + vehicle control (p.p.tid, 2wk) n=8

MCD飲食+己酮可可鹼(p.o.200mg/kg,tid,2wk) n=8 MCD diet + pentoxifylline (p.o.200mg/kg, tid, 2wk) n=8

MCD飲食+INT-747(p.o.30mg/kg,q.d.2wk) n=8 MCD diet+INT-747 (p.o.30mg/kg, q.d.2wk) n=8

MCD飲食+化合物1(p.o.30mg/kg,bid,2wk) n=8 MCD diet + compound 1 (p.o. 30mg/kg, bid, 2wk) n=8

給藥兩周後CO2處死小鼠，心臟取血500μL~600μL(K2-EDTA抗凝)，將血樣置於離心機中離心(4,500rpm,4℃,5分鐘)，分離上層得血漿， 分裝後速凍，保存在-80℃冰箱。並解剖取肝組織用於組織學分析與生物標志物檢測。 Two weeks after the administration, the mice were sacrificed with CO 2 and 500 μL~600 μL of blood was taken from the heart (K 2 -EDTA anticoagulation). The blood sample was centrifuged in a centrifuge (4,500 rpm, 4°C, 5 minutes), and the upper layer was separated to obtain plasma. Quickly freeze after sub-packaging and store in a refrigerator at -80°C. The liver tissue was dissected and used for histological analysis and biomarker detection.

凍存的血漿樣品於第二天進行ALT與AST檢測。 The frozen plasma samples were tested for ALT and AST the next day.

3.組織學分析方法。 3. Histological analysis methods.

3.1 HE染色方法。 3.1 HE staining method.

3.1.1組織處理。 3.1.1 Organizational processing.

使用Leica ASP200S全自動脫水機，相關的脫水步驟如下： Using Leica ASP200S automatic dehydration machine, the relevant dehydration steps are as follows:

a)10%中性緩衝福爾馬林5分鐘-6小時 a) 10% neutral buffered formalin for 5 minutes-6 hours

b)70%乙醇45分鐘 b) 70% ethanol for 45 minutes

c)80%乙醇45分鐘 c) 80% ethanol for 45 minutes

d)95%乙醇45分鐘 d) 95% ethanol for 45 minutes

e)95%乙醇45分鐘 e) 95% ethanol for 45 minutes

f)100%乙醇60分鐘 f) 100% ethanol for 60 minutes

g)100%乙醇60分鐘 g) 100% ethanol for 60 minutes

h)100%乙醇60分鐘 h) 100% ethanol for 60 minutes

i)二甲苯60分鐘 i) Xylene 60 minutes

j)二甲苯60分鐘 j) Xylene 60 minutes

k)石蠟60分鐘 k) Paraffin 60 minutes

l)石蠟60分鐘 l) Paraffin wax for 60 minutes

m)石蠟60分鐘 m) Paraffin 60 minutes

3.1.2組織包埋。 3.1.2 Tissue embedding.

a)包埋前的準備。 a) Preparation before embedding.

Leica組織包埋系統的組成及功能： Leica組織包埋系統由兩部分組成：包埋台(Leica EG1150 H)和冷凍台(Leica EG1150 C)。包埋台包括左側金屬模具存放缸，右側組織包埋盒存放缸及位於儀器上部的石蠟缸。包埋台前部有一個小的冷台，可以迅速冷却單個包埋模具。冷凍台主要由冷台構成，迅速冷却包埋模具內的石蠟。包埋盒冷却後，蠟塊保存在室溫條件下。 The composition and function of Leica tissue embedding system: Leica tissue embedding system consists of two parts: embedding table (Leica EG1150 H) and freezing table (Leica EG1150 C). The embedding table includes a metal mold storage cylinder on the left, a tissue embedding box storage cylinder on the right, and a paraffin wax cylinder on the upper part of the instrument. There is a small cold table at the front of the embedding table, which can quickly cool a single embedding mold. The freezing table is mainly composed of a cold table, which rapidly cools the paraffin in the embedding mold. After the cassette is cooled, the wax block is stored at room temperature.

b)打開冷台。 b) Open the cold table.

c)將脫水機中的包埋盒取出，放入包埋機蠟缸中。 c) Take out the embedding box from the dehydrator and put it into the wax bath of the embedding machine.

d)包埋。 d) Embedding.

將熱鑷子將包埋機蠟缸中的組織包埋盒取出，放在包埋機的熱臺上。將與組織相應的包埋盒放置在模具上面。用熱鑷子將組織放入包埋模具，再向包埋模具中加入少許溶解的石蠟，將包埋模具放在冷臺上，直至石蠟徹底變成固體狀態。最後將固體的蠟塊從包埋模具中取出。 Take the hot tweezers to take out the tissue embedding box in the wax bath of the embedding machine and place it on the hot stage of the embedding machine. Place the embedding cassette corresponding to the tissue on the mold. Put the tissue into the embedding mold with hot tweezers, then add a little dissolved paraffin to the embedding mold, and place the embedding mold on the cold table until the paraffin completely becomes a solid state. Finally, remove the solid wax block from the embedding mold.

3.1.3組織切片的準備。 3.1.3 Preparation of tissue sections.

a)使用Leica RM2235石蠟切片機前確認其狀態。 a) Confirm its status before using Leica RM2235 paraffin microtome.

裝蠟塊前，應確保切片輪把手粗語鎖定狀態，開啟刀架保護套，清潔刀架上的蠟屑。 Before loading the wax block, make sure that the handle of the slicing wheel is locked and open the protective cover of the knife holder to clean the wax chips on the knife holder.

小心插入未使用過的一次性切片刀，確認刀片放入到家中的適當位置，根據生產商的使用說明書鎖緊刀架。確認刀片的傾角，一般應設置為3-6度。 Carefully insert the unused disposable slicing knife, make sure that the blade is placed in the proper position at home, and lock the knife holder according to the manufacturer's instruction manual. Confirm the inclination angle of the blade, generally should be set to 3-6 degrees.

b)將組織蠟塊固定於切片機夾槽內的理想位置。 b) Fix the tissue wax block in the ideal position in the clip slot of the microtome.

根據生產商的使用說明書，使用粗修輪把手進行粗修，使蠟塊緩慢的向前推進，直至暴露所需的組織平面。 According to the manufacturer's instructions, use the rough trimming wheel handle to perform rough trimming, so that the wax block is slowly pushed forward until the desired tissue plane is exposed.

c)開始切片，首先開始設置切片厚度，開始切片，向前旋轉切片轉輪，直至平整的蠟片形成。鎖定切片輪轉把手，用鑷子或毛筆等將組織片 移入攤片機(Leica HI1210)並平整的裱於載玻片上。 c) Start slicing, first set the slice thickness, start slicing, and rotate the slicing wheel forward until a smooth wax slice is formed. Lock the slice rotation handle, and use tweezers or brush to remove the tissue Move it into the spreader (Leica HI1210) and mount it on a glass slide evenly.

d)完成粗修或切片後，鎖定輪轉把手，小心取出蠟塊。 d) After finishing rough trimming or sectioning, lock the rotary handle and carefully remove the wax block.

3.1.4 HE染色。 3.1.4 HE staining.

使用Leica ST5010、LeicaTS5010和LeicaCV5030組成的全自動染色工作站進行HE染色。 Use Leica ST5010, LeicaTS5010 and LeicaCV5030 composed of automatic dyeing workstation for HE dyeing.

在染色機主菜單中按<Stain>鍵。通過按鍵區的上下箭頭選擇程序15進行常規HE染色。打開裝載倉，放入切片架，關上裝載倉。按<Load>鍵後啟動染色程序。程序如下： Press the <Stain> key in the main menu of the dyeing machine. Use the up and down arrows in the keypad to select program 15 for conventional HE staining. Open the loading compartment, put in the slice rack, and close the loading compartment. Press the <Load> key to start the dyeing process. The procedure is as follows:

a)烤箱55℃ 2分鐘 a) Oven at 55℃ for 2 minutes

b)二甲苯I 10分鐘 b) Xylene I 10 minutes

c)二甲苯Ⅱ 10分鐘 c) Xylene Ⅱ 10 minutes

d)二甲苯Ⅲ 10分鐘 d) Xylene Ⅲ 10 minutes

e)無水乙醇I 2分鐘 e) Anhydrous ethanol I 2 minutes

f)無水乙醇Ⅱ 1.5分鐘 f) Anhydrous ethanol Ⅱ 1.5 minutes

g)95%乙醇I 1分鐘 g) 95% ethanol I for 1 minute

h)80%乙醇I 0.5分鐘 h) 80% ethanol I 0.5 minutes

i)水洗3分鐘 i) Wash in water for 3 minutes

j)蘇木素染液3分鐘 j) Hematoxylin dye solution for 3 minutes

k)水洗2分鐘 k) Wash in water for 2 minutes

l)1%鹽酸-乙醇5秒鐘 l) 1% hydrochloric acid-ethanol for 5 seconds

m)水洗2分鐘 m) Wash in water for 2 minutes

n)水洗20分鐘 n) Wash in water for 20 minutes

o)80%乙醇Ⅱ 1分鐘 o) 80% ethanolⅡ 1 minute

p)伊紅5秒鐘 p) Eosin 5 seconds

q)95%乙醇Ⅱ 30秒鐘 q) 95% ethanolⅡ 30 seconds

r)無水乙醇Ⅲ 2分鐘 r) Anhydrous ethanol Ⅲ 2 minutes

s)無水乙醇Ⅳ 2分鐘 s) Anhydrous ethanol IV 2 minutes

t)無水乙醇V 2分鐘 t) Anhydrous ethanol V 2 minutes

u)無水乙醇Ⅵ 2分鐘 u) Anhydrous ethanol Ⅵ 2 minutes

v)二甲苯Ⅳ 3分鐘 v) Xylene IV 3 minutes

w)二甲苯V 3分鐘 w) Xylene V 3 minutes

3.2組織學非酒精性脂肪肝病活動度評分(NAS)相關評分標準：非酒精性脂肪肝病(NAFLD)的組織學診斷和臨床療效評估參照美國國立衛生研究院NASH臨床研究網病理工作組指南，常規進行NAFLD活動度評分(NAFLD activity score,NAS)。 3.2 Histological non-alcoholic fatty liver disease activity score (NAS) related scoring standards: the histological diagnosis and clinical efficacy evaluation of non-alcoholic fatty liver disease (NAFLD) refer to the guidelines of the Pathology Working Group of the NASH Clinical Research Network of the National Institutes of Health, routine Perform NAFLD activity score (NAFLD activity score, NAS).

NAS評分(0~8分)： NAS score (0~8 points):

a)肝細胞脂肪變：0分(<5%)；1分(5%~33%)；2分(33%~66%)；3分(>66%)。 a) Hepatocyte steatosis: 0 points (<5%); 1 point (5%~33%); 2 points (33%~66%); 3 points (>66%).

b)小葉內炎症(20倍鏡計數壞死灶)：0分，無；1分(<2個)；2分(2~4個)；3分(>4個)。 b) Inflammation in the lobules (counting necrotic foci at 20 magnification): 0 points, none; 1 point (<2); 2 points (2 to 4); 3 points (>4).

c)肝細胞氣球樣變：0分，無；1分，少見；2分，多見。 c) Balloon change of liver cells: 0 points, none; 1 point, rare; 2 points, more common.

4．肝臟TG檢測方法。 4. Liver TG detection method.

4.1樣本準備：每份肝臟組織加入5% NP40裂解液，使得濃度為100mg/mL。在組織勻漿器上裂解組織。在80-100度水浴中加熱樣本5min，然後降至室溫。重複加熱一次。最高轉速離心2min，取上清。將上清稀釋60倍，檢測板中每孔加入30μL。 4.1 Sample preparation: Add 5% NP40 lysate to each liver tissue to make the concentration 100mg/mL. Lyse the tissue on a tissue homogenizer. Heat the sample in a water bath at 80-100 degrees for 5 minutes and then cool to room temperature. Repeat heating once. Centrifuge at the highest speed for 2 minutes, and take the supernatant. The supernatant was diluted 60 times, and 30 μL was added to each well of the detection plate.

4.2標準品準備：將1mM甘油三酯稀釋至0.2mM，在標準孔列分別加入0,10,20,30,40,50μL的0.2mM甘油三酯標準品，加檢測液補足到50μL。 4.2 Standard preparation: Dilute 1 mM triglyceride to 0.2 mM, add 0, 10, 20, 30, 40, 50 μL of 0.2 mM triglyceride standard to the standard wells, and add test solution to make up to 50 μL.

4.3每孔加入2μL脂肪酶，混勻室溫孵育20min。 4.3 Add 2μL lipase to each well, mix well and incubate at room temperature for 20min.

4.4按比例配置酶反應液：46μL甘油三酯檢測液+2μL甘油三酯探針+2μL甘油三酯酶反應液。向所有檢測孔中加入50μL酶反應液，混勻，避光室溫孵育30-60min。 4.4 Prepare the enzyme reaction solution in proportion: 46μL triglyceride detection solution + 2μL triglyceride probe + 2μL triglyceride enzyme reaction solution. Add 50μL of enzyme reaction solution to all detection wells, mix well, and incubate at room temperature in the dark for 30-60min.

4.5檢測：用酶標儀測定570nm的吸光度值。 4.5 Detection: Measure the absorbance value at 570nm with a microplate reader.

5.肝組織TNF-αmRNA檢測方法。 5. Detection method of TNF-αmRNA in liver tissue.

根據操作手册，用RNeasy® Mini RNA提取試劑盒(Qiagen #74104)提取RNA，當天完成逆轉錄，逆轉錄體系配比(ABI # 4368814)如下：

Figure 105135042-A0305-02-0175-165
According to the operation manual, RNA was extracted with RNeasy® Mini RNA Extraction Kit (Qiagen #74104), and reverse transcription was completed on the same day. The ratio of the reverse transcription system (ABI # 4368814) is as follows:
Figure 105135042-A0305-02-0175-165

逆轉錄程序： Reverse transcription program:

Figure 105135042-A0305-02-0175-164
Figure 105135042-A0305-02-0175-164

cDNA保存於-20℃冰箱。 cDNA is stored in a refrigerator at -20°C.

按照TaqMan® Fast Universal試劑盒(ABI,# 4366072)說明書，進行Q-PCR檢測，體系配比如下：TaqMan® Gene Expression Master Mix(2×) 5μL

Figure 105135042-A0305-02-0176-167
Follow the instructions of TaqMan® Fast Universal Kit (ABI, # 4366072) to perform Q-PCR detection. The system configuration is as follows: TaqMan® Gene Expression Master Mix (2×) 5μL
Figure 105135042-A0305-02-0176-167

Q-PCR程序： Q-PCR program:

Figure 105135042-A0305-02-0176-166
Figure 105135042-A0305-02-0176-166

6.炎症因子檢測方法。 6. Detection methods of inflammatory factors.

6.1肝組織勻漿與樣品製備。 6.1 Homogenization of liver tissue and sample preparation.

6.1.1開機預冷離心機。 6.1.1 Start the pre-cooling centrifuge.

6.1.2將一片One cOmplete,EDTA-free protease inhibitor tablet溶於50mL磷酸鹽緩衝液中，PBS置於濕冰中均漿1：10(w/v)。 6.1.2 Dissolve a piece of One cOmplete, EDTA-free protease inhibitor tablet in 50 mL of phosphate buffer, and place PBS on wet ice to homogenize 1:10 (w/v).

6.1.3離心1000g,10min,4℃，上清轉移220μL至超速離心管中，每份上清準備兩管超速離心。 6.1.3 Centrifuge at 1000g, 10min, 4℃, transfer 220μL of the supernatant to an ultracentrifuge tube, prepare two ultracentrifugation tubes for each supernatant.

6.1.4樣品置於已經預冷的超速離心機OptimaTM L-80 XP(Beckman)中，超速離心100,000g，4℃，50min。 6.1.4 The sample was placed in a pre-cooled ultracentrifuge Optima TM L-80 XP (Beckman), and ultracentrifuged at 100,000 g at 4°C for 50 min.

6.1.5乾冰上預冷V底96孔板，將同一樣品對應的兩管上清並入1.5mL EP管中，輕彈混勻後取120μL分裝至V底96孔板中，乾冰速凍，共製備三塊樣品板(一塊板用於BCA蛋白定量，一塊板用於MSD檢測，剩餘一塊備用)。 6.1.5 Pre-cool the V-bottom 96-well plate on dry ice, merge the two tubes of supernatant corresponding to the same sample into a 1.5mL EP tube, flick and mix, take 120μL aliquot into the V-bottom 96-well plate, and quickly freeze on dry ice. A total of three sample plates are prepared (one plate is used for BCA protein quantification, one plate is used for MSD detection, and the remaining one is for use).

6.2 BCA法測定組織勻漿液中蛋白濃度。 6.2 BCA method to determine the protein concentration in the tissue homogenate.

6.2.1 BCA工作液製備：按照試劑A：試劑B=50：1混合試劑A與B。 6.2.1 Preparation of BCA working solution: mix Reagent A and B according to Reagent A: Reagent B=50:1.

6.2.2製備BSA(牛血清白蛋)標準溶液A~I。 6.2.2 Prepare BSA (bovine serum albumin) standard solutions A~I.

Figure 105135042-A0305-02-0177-168
Figure 105135042-A0305-02-0177-168

6.2.3用PBS稀釋勻漿液，20μL勻漿液+80μL PBS。 6.2.3 Dilute the homogenate with PBS, 20μL homogenate + 80μL PBS.

6.2.4在96孔板中按順序加入10μL BSA標準液A~I及已稀釋的待測樣品。 6.2.4 Add 10μL of BSA standard solution A~I and the diluted sample to be tested in order in the 96-well plate.

6.2.5每孔加入200μLBCA工作液於標準液和待測樣品中，振板器上混勻。 6.2.5 Add 200μLBCA working solution to each well of the standard solution and the sample to be tested, and mix on the shaker.

6.2.6在37℃孵育箱中孵育30分鐘。 6.2.6 Incubate in a 37°C incubator for 30 minutes.

6.2.7取出板後放冷至室溫。 6.2.7 After taking out the plate, let it cool to room temperature.

6.2.8在酶標儀上用562nm波長測吸光度。 6.2.8 Use the 562nm wavelength to measure the absorbance on the microplate reader.

6.3 MSD檢測樣品中炎症因子含量。 6.3 MSD detects the content of inflammatory factors in the sample.

6.3.1冰箱中取出MSD板，放置於室溫。 6.3.1 Take out the MSD plate from the refrigerator and place it at room temperature.

6.3.2按照1：3梯度稀釋標準品溶液。 6.3.2 Dilute the standard solution in a 1:3 gradient.

6.3.3每孔加入50μL標準品與樣品溶液，固定於振板上，300-1000rpm室溫孵育2hr。 6.3.3 Add 50μL of standard and sample solution to each well, fix it on a vibrating plate, and incubate at 300-1000rpm for 2hr at room temperature.

6.3.4每孔加入300μLWash buffer洗板三次，每孔加入1×檢測抗體溶液25μL，固定於振板上，300-1000rpm室溫孵育2hr。 6.3.4 Add 300μL Wash buffer to each well to wash the plate three times, add 25μL of 1× detection antibody solution to each well, fix it on the shaker plate, and incubate at 300-1000rpm for 2hr at room temperature.

6.3.5每孔加入300μL Wash buffer洗板三次，每孔加入1×讀板液150μL，放入Sector Imager 6000 Model 1200中讀取信號值。 6.3.5 Add 300μL Wash buffer to each well to wash the plate three times, add 1×150μL of plate reading solution to each well, and put it into Sector Imager 6000 Model 1200 to read the signal value.

實驗結果：。 Experimental results:

1.組織學結果。 1. Histological results.

1.1 HE染色圖片。 1.1 HE stained picture.

HE染色結果顯示，MCD飼養四周，小鼠肝臟脂肪變顯著增加，肝小葉炎症明顯增加；己酮可可鹼(200mpk,po.,t.i.d.)與INT-747(30mpk,po.,q.d.)給藥兩周，MCD小鼠肝臟病變並無顯著改善，而化合物1(30mpk,po.,b.i.d.)給藥兩周MCD小鼠肝小葉炎症明顯改善，實驗結果見圖1。 The results of HE staining showed that the mice were fed with MCD for four weeks, and liver fat became significantly increased, and liver lobule inflammation was significantly increased. Pentoxifylline (200mpk, po., tid) and INT-747 (30mpk, po., qd) were administered twice. In weeks, the liver lesions of MCD mice did not significantly improve, while the liver lobule inflammation of MCD mice was significantly improved after compound 1 (30mpk, po., bid) was administered for two weeks. The experimental results are shown in Figure 1.

1.2 NAS評分。 1.2 NAS score.

MCD小鼠肝臟組織學NAS和小葉炎症顯著升高，本次實驗測試的三種化合物對NAS無顯著性影響，但化合物1能夠顯著性降低MCD小鼠肝臟小葉炎症，實驗結果見圖2。 The histology of liver NAS and lobular inflammation in MCD mice were significantly increased. The three compounds tested in this experiment had no significant effect on NAS, but compound 1 could significantly reduce liver lobular inflammation in MCD mice. The experimental results are shown in Figure 2.

2．肝臟TG檢測結果。 2. Liver TG test results.

MCD小鼠肝臟TG水平顯著升高，本次實驗測試的三種化合物對MCD小鼠肝臟TG水平均無顯著性影響，實驗結果見圖3。 The liver TG level of MCD mice was significantly increased. The three compounds tested in this experiment had no significant effect on the liver TG level of MCD mice. The experimental results are shown in Figure 3.

3.肝臟TNF-αmRNA水平檢測結果。 3. Results of detection of liver TNF-αmRNA levels.

化合物1(30mpk,po.,b.i.d.)給藥兩周MCD小鼠肝臟TNF-αmRNA水平有下降趨勢，但經統計學比較沒有顯著性差異，實驗結果見圖4。 Compound 1 (30mpk, po., b.i.d.) was administered for two weeks. The liver TNF-α mRNA level of MCD mice showed a downward trend, but there was no significant difference after statistical comparison. The experimental results are shown in Figure 4.

4．肝臟炎症因子檢測結果。 4. Test results of liver inflammatory factors.

MCD飼養4周，小鼠肝臟IL-1β蛋白水平明顯升高，己酮可可鹼(200mpk,po.,t.i.d.)與INT-747(30mpk,po.,q.d.)給藥兩周對MCD小鼠肝臟 IL-1β蛋白水平沒有顯著性影響；化合物1(30mpk,po.,b.i.d.)給藥兩周MCD小鼠肝臟IL-1β蛋白水平顯著下降，實驗結果見圖5。 After 4 weeks of MCD feeding, the level of IL-1β protein in the liver of mice was significantly increased. Pentoxifylline (200mpk, po., t.i.d.) and INT-747 (30mpk, po., q.d.) were administered to the liver of MCD mice for two weeks. IL-1β protein level has no significant effect; compound 1 (30mpk, po., b.i.d.) administration of MCD mice liver IL-1β protein level significantly decreased for two weeks, the experimental results are shown in Figure 5.

5．血漿炎症因子檢測結果。 5. Test results of plasma inflammatory factors.

MCD小鼠血漿TNF-α與IL-1β蛋白水平顯著升高，己酮可可鹼(200mpk,po.,t.i.d.)與INT-747(30mpk,po.,q.d.)給藥兩周對MCD小鼠血漿TNF-α與IL-1β蛋白水平無顯著性影響，化合物1(30mpk,po.,b.i.d.)給藥兩周MCD小鼠血漿TNF-α與IL-1β蛋白水平顯著下降，實驗結果見圖6。 The plasma TNF-α and IL-1β protein levels of MCD mice were significantly increased. Pentoxifylline (200mpk, po., tid) and INT-747 (30mpk, po., qd) were administered to the plasma of MCD mice for two weeks. The protein levels of TNF-α and IL-1β had no significant effect. The plasma TNF-α and IL-1β protein levels of MCD mice were significantly decreased after compound 1 (30mpk, po., bid) was administered for two weeks. The experimental results are shown in Figure 6.

6.血漿ALT與AST檢測結果。 6. Plasma ALT and AST test results.

MCD小鼠血漿ALT與AST水平顯著升高，本次實驗測試的三種化合物均不能降低MCD小鼠血漿ALT與AST水平，實驗結果見圖7。 The plasma ALT and AST levels of MCD mice were significantly increased. None of the three compounds tested in this experiment could reduce the plasma ALT and AST levels of MCD mice. The experimental results are shown in Figure 7.

總結。 to sum up.

1.MCD小鼠是廣泛使用的非酒精性脂肪性肝炎動物模型，模型小鼠肝臟脂肪變性明顯，TG水平顯著升高；組織學HE染色圖片顯示出明顯的小葉炎症，肝臟TNF-αmRNA顯著升高，肝臟炎症因子IL-1β，血漿TNF-α和IL-1β的蛋白水平均顯著高於正常；血漿ALT與AST水平較正常小鼠顯著升高。 1. MCD mice are a widely used animal model of non-alcoholic steatohepatitis. The model mice have obvious liver steatosis, and the TG level is significantly increased; the histological HE staining picture shows obvious lobular inflammation, and the liver TNF-αmRNA is significantly increased. High, liver inflammation factor IL-1β, plasma TNF-α and IL-1β protein levels were significantly higher than normal; plasma ALT and AST levels were significantly higher than normal mice.

2.化合物1(30mpk,po.,b.i.d.)兩周給藥後，MCD小鼠肝臟小葉炎症顯著改善，肝臟炎症因子IL-1β，血漿TNF-α和IL-1β蛋白水平均顯著下降。 2. After two weeks of administration of compound 1 (30mpk, po., b.i.d.), the inflammation of liver lobules in MCD mice was significantly improved, and the liver inflammatory factor IL-1β, plasma TNF-α and IL-1β protein levels were significantly reduced.

3.化合物1(30mpk,po.,b.i.d.)兩周給藥，對MCD小鼠肝臟TG水平，肝臟TNF-αmRNA，血漿ALT與AST水平無顯著性影響。 3. Compound 1 (30mpk, po., b.i.d.) administered for two weeks has no significant effect on liver TG levels, liver TNF-αmRNA, plasma ALT and AST levels in MCD mice.

實驗例5nSTZ+HFD小鼠模型體內藥理學研究。 Experimental Example 5 In vivo pharmacological study of nSTZ+HFD mouse model.

主要任務。 main mission.

評估化合物1對非酒精性脂肪性肝炎模型nSTZ+HFD小鼠肝臟組織學，TNF-αmRNA，TGF-β1 mRNA和collagen 1α1 mRNA水平，血清ALT，AST，TG和TC水平的影響。 To evaluate the effect of compound 1 on liver histology, TNF-αmRNA, TGF-β1 mRNA and collagen 1α1 mRNA levels, serum ALT, AST, TG and TC levels in the nSTZ+HFD mouse model of non-alcoholic steatohepatitis.

擬解决的問題。 The problem to be solved.

測試化合物1對nSTZ+HFD小鼠肝臟組織學，TNF-αmRNA，TGF-β1 mRNA和Collagen 1α1 mRNA水平，血清ALT，AST，TG和TC水平的影響，評估化合物對非酒精性脂肪性肝炎模型小鼠的肝功能保護，降低肝臟炎症和纖維化的作用。 Test the effect of compound 1 on liver histology, TNF-αmRNA, TGF-β1 mRNA and Collagen 1α1 mRNA levels, serum ALT, AST, TG and TC levels in nSTZ+HFD mice, and evaluate the compound's effect on the model of non-alcoholic steatohepatitis. Protect the liver function of mice, reduce liver inflammation and fibrosis.

實驗模型：新生小鼠STZ注射合併高脂飲食誘導的非酒精性脂肪性肝炎模型(nSTZ+HFD mice)。 Experimental model: Neonatal mice STZ injection combined with high-fat diet induced non-alcoholic steatohepatitis model (nSTZ+HFD mice).

實驗動物：C57BL/6J小鼠(雌性孕鼠，孕14-15天購入，上海靈暢生物科技有限公司)。 Experimental animals: C57BL/6J mice (female pregnant mice, purchased at 14-15 days gestation, Shanghai Lingchang Biotechnology Co., Ltd.).

實驗試劑： Experimental reagents:

1.鏈脲黴素(Streptozocin，STZ)(Sigma,#S0130-1G) 1. Streptozocin (STZ) (Sigma, #S0130-1G)

2.高脂飼料(Research Diets,# D12492i) 2. High-fat feed (Research Diets, # D12492i)

3.RNeasy® Mini RNA提取試劑盒(Qiagen,#74104) 3.RNeasy® Mini RNA extraction kit (Qiagen, #74104)

4.High Capacity cDNA逆轉錄試劑盒(ABI,# 4368814) 4. High Capacity cDNA Reverse Transcription Kit (ABI, # 4368814)

5.TaqMan® Gene Expression Assay TNF-α(ABI,Assay ID：Mm00443258_m1) 5. TaqMan® Gene Expression Assay TNF-α (ABI, Assay ID: Mm00443258_m1)

6.TaqMan® Gene Expression Assay TGF-β1(ABI,Assay ID：Mm00441724_m1) 6. TaqMan® Gene Expression Assay TGF-β1 (ABI, Assay ID: Mm00441724_m1)

7.TaqMan® Gene Expression Assay Collagen 1α1(ABI,Assay ID：Mm00801666_g1) 7. TaqMan® Gene Expression Assay Collagen 1α1 (ABI, Assay ID: Mm00801666_g1)

8.TaqMan® Endogenous Control 18S(ABI,Assay ID：Mm03928990_g1) 8. TaqMan® Endogenous Control 18S (ABI, Assay ID: Mm03928990_g1)

9.TaqMan® Fast Universal PCR Mater Mix(ABI,# 4366072) 9.TaqMan® Fast Universal PCR Mater Mix(ABI,# 4366072)

10.Hematoxylin(Baso,# BA4097) 10.Hematoxylin(Baso,# BA4097)

11.Eosin(Baso,# BA4099) 11.Eosin(Baso,# BA4099)

12.Sirius red(Bogoo,# PT036) 12.Sirius red(Bogoo,#PT036)

實驗方法。 experimental method.

1.實驗流程。 1. Experimental process.

Figure 105135042-A0305-02-0181-170
Figure 105135042-A0305-02-0181-170

2.體內實驗方法。 2. In vivo experimental methods.

購買14~15天孕齡C57BL/6J小鼠，新生鼠出生2~3天進行STZ注射(200ug,s.c.)。普通飼料飼養至4周齡開始給予高脂飲食。高脂飼養1周後動物隨機分組，開始口服給藥。 C57BL/6J mice with a gestational age of 14 to 15 days were purchased, and the newborn mice were injected with STZ (200ug, s.c.) 2 to 3 days after birth. The normal feed was raised to 4 weeks of age and started to be given a high-fat diet. After one week of high-fat feeding, the animals were randomly divided into groups and started oral administration.

分組與給藥信息如下：對照組(STZ溶媒對照+正常飲食) n=12 Grouping and administration information is as follows: control group (STZ vehicle control + normal diet) n=12

模型組(STZ+高脂飲食)+溶媒對照(0.5% HPMC,p.o.t.i.d.) n=20 Model group (STZ + high fat diet) + vehicle control (0.5% HPMC, p.o.t.i.d.) n=20

模型組(STZ+高脂飲食)+己酮可可鹼(100mg/kg,p.o,t.i.d.) n=20 Model group (STZ+high-fat diet)+Pentoxifylline (100mg/kg, p.o, t.i.d.) n=20

模型組(STZ+高脂飲食)+化合物1(30mg/kg,p.o,b.i.d.) n=19連續給藥5周後過夜禁食。CO2處死小鼠，心臟取血後室溫放置2小時，將血樣置於離心機中離心(2000g,4℃,15分鐘)，分離上層得血清，分裝後速凍，保存在-80℃冰箱。並解剖取肝組織用於組織學分析與生物標志物檢測。 Model group (STZ+high-fat diet)+Compound 1 (30mg/kg, po, bid) n=19 after continuous administration for 5 weeks and overnight fasting. The mice were killed by CO 2 and the heart was collected and placed at room temperature for 2 hours. The blood sample was centrifuged in a centrifuge (2000g, 4℃, 15 minutes), and the upper layer was separated to obtain the serum, aliquoted and quick-frozen, and stored in a -80℃ refrigerator . The liver tissue was dissected and used for histological analysis and biomarker detection.

凍存的血清樣品於第二天進行ALT，AST，TG和TC檢測。 The frozen serum samples were tested for ALT, AST, TG and TC the next day.

3．組織學分析方法。 3. Histological analysis methods.

3.1 HE染色方法。 3.1 HE staining method.

3.1.1組織處理。 3.1.1 Organizational processing.

使用Leica ASP200S全自動脫水機，相關的脫水步驟如下： Using Leica ASP200S automatic dehydration machine, the relevant dehydration steps are as follows:

a)10%中性緩衝福爾馬林5分鐘~6小時 a) 10% neutral buffered formalin for 5 minutes to 6 hours

b)70%乙醇45分鐘 b) 70% ethanol for 45 minutes

c)80%乙醇45分鐘 c) 80% ethanol for 45 minutes

d)95%乙醇45分鐘 d) 95% ethanol for 45 minutes

e)95%乙醇45分鐘 e) 95% ethanol for 45 minutes

f)100%乙醇60分鐘 f) 100% ethanol for 60 minutes

g)100%乙醇60分鐘 g) 100% ethanol for 60 minutes

h)100%乙醇60分鐘 h) 100% ethanol for 60 minutes

i)二甲苯60分鐘 i) Xylene 60 minutes

j)二甲苯60分鐘 j) Xylene 60 minutes

k)石蠟60分鐘 k) Paraffin 60 minutes

l)石蠟60分鐘 l) Paraffin wax for 60 minutes

m)石蠟60分鐘 m) Paraffin 60 minutes

3.1.2組織包埋。 3.1.2 Tissue embedding.

a)包埋前的準備。 a) Preparation before embedding.

Leica組織包埋系統的組成及功能：Leica組織包埋系統由兩部分組成：包埋台(Leica EG1150 H)和冷凍台(Leica EG1150 C)。包埋台包括左側金屬模具存放缸，右側組織包埋盒存放缸及位於儀器上部的石蠟缸。包埋台前部有一個小的冷台，可以迅速冷却單個包埋模具。冷凍台主要由冷台構成，迅速冷却包埋模具內的石蠟。包埋盒冷却後，蠟塊保存在室溫條件下。 The composition and function of Leica tissue embedding system: Leica tissue embedding system consists of two parts: embedding table (Leica EG1150 H) and freezing table (Leica EG1150 C). The embedding table includes a metal mold storage cylinder on the left, a tissue embedding box storage cylinder on the right, and a paraffin wax cylinder on the upper part of the instrument. There is a small cold table at the front of the embedding table, which can quickly cool a single embedding mold. The freezing table is mainly composed of a cold table, which rapidly cools the paraffin in the embedding mold. After the cassette is cooled, the wax block is stored at room temperature.

b)打開冷台。 b) Open the cold table.

c)將脫水機中的包埋盒取出，放入包埋機蠟缸中 c) Take out the embedding box from the dehydrator and put it in the wax bath of the embedding machine

d)包埋。 d) Embedding.

將熱鑷子將包埋機蠟缸中的組織包埋盒取出，放在包埋機的熱臺上。將與組織相應的包埋盒放置在模具上面。用熱鑷子將組織放入包埋模具，再向包埋模具中加入少許溶解的石蠟，將包埋模具放在冷臺上，直至石蠟徹底變成固體狀態。最後將固體的蠟塊從包埋模具中取出。 Take the hot tweezers to take out the tissue embedding box in the wax bath of the embedding machine and place it on the hot stage of the embedding machine. Place the embedding cassette corresponding to the tissue on the mold. Put the tissue into the embedding mold with hot tweezers, then add a little dissolved paraffin to the embedding mold, and place the embedding mold on the cold table until the paraffin completely becomes a solid state. Finally, remove the solid wax block from the embedding mold.

3.1.3組織切片的準備。 3.1.3 Preparation of tissue sections.

a)使用Leica RM2235石蠟切片機前確認其狀態。 a) Confirm its status before using Leica RM2235 paraffin microtome.

裝蠟塊前，應確保切片輪把手粗語鎖定狀態，開啟刀架保護套，清潔刀架上的蠟屑。 Before loading the wax block, make sure that the handle of the slicing wheel is locked and open the protective cover of the knife holder to clean the wax chips on the knife holder.

小心插入未使用過的一次性切片刀，確認刀片放入到家中的適當位置，根據生產商的使用說明書鎖緊刀架。確認刀片的傾角，一般應設置為3-6度。 Carefully insert the unused disposable slicing knife, make sure that the blade is placed in the proper position at home, and lock the knife holder according to the manufacturer's instruction manual. Confirm the inclination angle of the blade, generally should be set to 3-6 degrees.

b)將組織蠟塊固定於切片機夾槽內的理想位置。 b) Fix the tissue wax block in the ideal position in the clip slot of the microtome.

根據生產商的使用說明書，使用粗修輪把手進行粗修，使蠟塊緩慢的向前推進，直至暴露所需的組織平面。 According to the manufacturer's instructions, use the rough trimming wheel handle to perform rough trimming, so that the wax block is slowly pushed forward until the desired tissue plane is exposed.

c)開始切片，首先開始設置切片厚度，開始切片，向前旋轉切片轉輪，直至平整的蠟片形成。鎖定切片輪轉把手，用鑷子或毛筆等將組織片移入攤片機(Leica HI1210)並平整的裱於載玻片上。 c) Start slicing, first set the slice thickness, start slicing, and rotate the slicing wheel forward until a smooth wax slice is formed. Lock the rotary handle of the section, use tweezers or a brush to move the tissue piece into the spreader (Leica HI1210) and mount it on the glass slide evenly.

d)完成粗修或切片後，鎖定輪轉把手，小心取出蠟塊。 d) After finishing rough trimming or sectioning, lock the rotary handle and carefully remove the wax block.

3.1.4 HE染色。 3.1.4 HE staining.

使用Leica ST5010、LeicaTS5010和LeicaCV5030組成的全自動染色工作站進行HE染色。 Use Leica ST5010, LeicaTS5010 and LeicaCV5030 composed of automatic dyeing workstation for HE dyeing.

在染色機主菜單中按<Stain>鍵。通過按鍵區的上下箭頭選擇程序15進行常規HE染色。打開裝載倉，放入切片架，關上裝載倉。按<Load>鍵後啟動染色程序。程序如下： Press the <Stain> key in the main menu of the dyeing machine. Use the up and down arrows in the keypad to select program 15 for conventional HE staining. Open the loading compartment, put in the slice rack, and close the loading compartment. Press the <Load> key to start the dyeing process. The procedure is as follows:

a)烤箱55℃ 2分鐘 a) Oven at 55℃ for 2 minutes

b)二甲苯I 10分鐘 b) Xylene I 10 minutes

c)二甲苯Ⅱ 10分鐘 c) Xylene Ⅱ 10 minutes

d)二甲苯Ⅲ 10分鐘 d) Xylene Ⅲ 10 minutes

e)無水乙醇I 2分鐘 e) Anhydrous ethanol I 2 minutes

f)無水乙醇Ⅱ 1.5分鐘 f) Anhydrous ethanol Ⅱ 1.5 minutes

g)95%乙醇I 1分鐘 g) 95% ethanol I for 1 minute

h)80%乙醇I 0.5分鐘 h) 80% ethanol I 0.5 minutes

i)水洗3分鐘 i) Wash in water for 3 minutes

j)蘇木素染液3分鐘 j) Hematoxylin dye solution for 3 minutes

k)水洗2分鐘 k) Wash in water for 2 minutes

l)1%鹽酸-乙醇5秒鐘 l) 1% hydrochloric acid-ethanol for 5 seconds

m)水洗2分鐘 m) Wash in water for 2 minutes

n)水洗20分鐘 n) Wash in water for 20 minutes

o)80%乙醇Ⅱ 1分鐘 o) 80% ethanolⅡ 1 minute

p)伊紅5秒鐘 p) Eosin 5 seconds

q)95%乙醇Ⅱ 30秒鐘 q) 95% ethanolⅡ 30 seconds

r)無水乙醇Ⅲ 2分鐘 r) Anhydrous ethanol Ⅲ 2 minutes

s)無水乙醇Ⅳ 2分鐘 s) Anhydrous ethanol IV 2 minutes

t)無水乙醇V 2分鐘 t) Anhydrous ethanol V 2 minutes

u)無水乙醇Ⅵ 2分鐘 u) Anhydrous ethanol Ⅵ 2 minutes

v)二甲苯Ⅳ 3分鐘 v) Xylene IV 3 minutes

w)二甲苯V 3分鐘 w) Xylene V 3 minutes

x)二甲苯V 3分鐘 x) Xylene V 3 minutes

y)EXIT二甲苯3分鐘 y) EXIT xylene 3 minutes

z)Leica CV5030全自動封片機封蓋切片 z) Leica CV5030 Fully Automatic Covering Machine for Capping and Slicing

3.2天狼星紅染色方法。 3.2 Sirius red staining method.

將3.1.3製備好的組織白片通過手工製作天狼星紅染色切片，步驟如下： The white tissue slice prepared in 3.1.3 is hand-made into Sirius red stained sections, the steps are as follows:

a)二甲苯I 15分鐘 a) Xylene I 15 minutes

b)二甲苯Ⅱ 15分鐘 b) Xylene Ⅱ 15 minutes

c)無水乙醇I 3分鐘 c) Anhydrous ethanol I 3 minutes

d)無水乙醇Ⅱ 3分鐘 d) Anhydrous ethanol Ⅱ 3 minutes

e)95%乙醇I 3分鐘 e) 95% ethanol I 3 minutes

f)95%乙醇Ⅱ 3分鐘 f) 95% ethanolⅡ 3 minutes

g)80%乙醇3分鐘 g) 80% ethanol for 3 minutes

h)70%乙醇3分鐘 h) 70% ethanol for 3 minutes

i)蒸餾水I 3分鐘 i) Distilled water I 3 minutes

j)蒸餾水Ⅱ 3分鐘 j) Distilled water Ⅱ 3 minutes

k)蒸餾水Ⅲ 3分鐘 k) Distilled water Ⅲ 3 minutes

l)天狼星紅染液1小時 l) Sirius red dye solution for 1 hour

m)蒸餾水5分鐘 m) Distilled water for 5 minutes

n)95%乙醇Ⅲ 30秒鐘 n) 95% ethanolⅢ 30 seconds

o)無水乙醇Ⅲ 2分鐘 o) Anhydrous ethanol Ⅲ 2 minutes

p)無水乙醇Ⅳ 2分鐘 p) Anhydrous ethanol IV 2 minutes

q)無水乙醇V 2分鐘 q) Anhydrous ethanol V 2 minutes

r)無水乙醇Ⅵ 2分鐘 r) Anhydrous ethanol Ⅵ 2 minutes

s)二甲苯Ⅳ 15分鐘 s) Xylene IV 15 minutes

t)二甲苯V 15分鐘 t) Xylene V 15 minutes

u)中性樹膠封片 u) Neutral gum seal

3.3組織學非酒精性脂肪肝病活動度評分(NAS)相關評分標準。 3.3 Histological non-alcoholic fatty liver disease activity score (NAS) related scoring standards.

非酒精性脂肪肝病(NAFLD)的組織學診斷和臨床療效評估參照美國國立衛生研究院NASH臨床研究網病理工作組指南，常規進行NAFLD活動度評分(NAFLD activity score,NAS)。 The histological diagnosis and clinical efficacy evaluation of non-alcoholic fatty liver disease (NAFLD) refer to the guidelines of the Pathology Working Group of the National Institutes of Health NASH Clinical Research Network, and NAFLD activity score (NAS) is routinely performed.

NAS評分(0~8分)： NAS score (0~8 points):

a)肝細胞脂肪變：0分(<5%)；1分(5%~33%)；2分(33%~66%)；3分(>66%)。 a) Hepatocyte steatosis: 0 points (<5%); 1 point (5%~33%); 2 points (33%~66%); 3 points (>66%).

b)小葉內炎症(20倍鏡計數壞死灶)：0分，無；1分(<2個)；2分(2~4個)；3分(>4個)。 b) Inflammation in the lobules (counting necrotic foci at 20 magnification): 0 points, none; 1 point (<2); 2 points (2 to 4); 3 points (>4).

c)肝細胞氣球樣變：0分，無；1分，少見；2分，多見。 c) Balloon change of liver cells: 0 points, none; 1 point, rare; 2 points, more common.

3.4纖維化評分標準。 3.4 Fibrosis scoring standard.

肝纖維化分期(0~4)： Stages of liver fibrosis (0~4):

a)0分：無纖維化； a) 0 points: no fibrosis;

b)1分：1A，肝腺泡3區輕度竇周纖維化；1B，肝腺泡3區中度竇周纖維化；1C，僅有門脉周圍纖維化； b) 1 point: 1A, mild perisinus fibrosis in liver acinar zone 3; 1B, moderate perisinus fibrosis in liver acinar zone 3; 1C, only periportal fibrosis;

c)2分：肝腺泡3區竇周纖維化合併門脉周圍纖維化； c) 2 points: perisinus fibrosis in area 3 of liver acinus combined with periportal fibrosis;

d)3分：橋接纖維化；以及 d) 3 points: bridging fibrosis; and

e)4分：高度可疑或確診肝硬化。 e) 4 points: highly suspicious or confirmed liver cirrhosis.

4.肝組織生物標志物mRNA檢測方法。 4. Detection method of liver tissue biomarker mRNA.

根據操作手册，用RNeasy® Mini RNA提取試劑盒(Qiagen #74104)提取RNA，當天完成逆轉錄，逆轉錄體系配比(ABI # 4368814)如下：

Figure 105135042-A0305-02-0187-173
According to the operation manual, RNA was extracted with RNeasy® Mini RNA Extraction Kit (Qiagen #74104), and reverse transcription was completed on the same day. The ratio of the reverse transcription system (ABI # 4368814) is as follows:
Figure 105135042-A0305-02-0187-173

逆轉錄程序： Reverse transcription program:

Figure 105135042-A0305-02-0187-171
Figure 105135042-A0305-02-0187-171

cDNA保存於-20℃冰箱。 cDNA is stored in a refrigerator at -20°C.

按照TaqMan® Fast Universal試劑盒(ABI,# 4366072)說明書，進行Q-PCR檢測，體系配比如下：

Figure 105135042-A0305-02-0187-236
Follow the instructions of TaqMan® Fast Universal Kit (ABI, # 4366072) to perform Q-PCR detection. The system configuration is as follows:
Figure 105135042-A0305-02-0187-236

Q-PCR程序： Q-PCR program:

Figure 105135042-A0305-02-0187-172
Figure 105135042-A0305-02-0187-172
Figure 105135042-A0305-02-0188-174
Figure 105135042-A0305-02-0188-174

實驗結果。 Experimental results.

1.組織學結果。 1. Histological results.

1.1 HE染色圖片。 1.1 HE stained picture.

HE染色結果顯示，nSTZ+HFD小鼠肝臟脂肪變性顯著增加，肝細胞出現氣球樣變，肝小葉炎症明顯增加，己酮可可鹼(100mpk,po.,t.i.d.)與化合物1(30mpk,po.,b.i.d.)5周治療並不能顯著改善nSTZ+HFD小鼠肝臟組織脂肪樣變、氣球樣變性和炎症聚集的病理性改變。實驗結果見圖8。 HE staining results showed that nSTZ+HFD mice showed a significant increase in liver steatosis, balloon-like changes in liver cells, and a significant increase in inflammation of liver lobules. Pentoxifylline (100mpk, po., tid) and compound 1 (30mpk, po., bid) 5 weeks of treatment did not significantly improve the pathological changes of steatosis, ballooning and inflammation accumulation in the liver tissue of nSTZ+HFD mice. The experimental results are shown in Figure 8.

1.2 NAS評分。 1.2 NAS score.

nSTZ+HFD小鼠肝臟組織學NAS顯著升高，肝臟脂肪化顯著升高，少量肝細胞氣球樣變，肝小葉炎症顯著增加。己酮可可鹼(100mpk,po.,t.i.d.)與化合物1(30mpk,po.,b.i.d.)5周治療並不能顯著影響nSTZ+HFD小鼠的肝臟NAS。實驗結果見圖9。 In nSTZ+HFD mice, liver histology NAS was significantly increased, liver fatification was significantly increased, a small amount of hepatocytes ballooned, and liver lobule inflammation was significantly increased. Pentoxifylline (100mpk, po., t.i.d.) and compound 1 (30mpk, po., b.i.d.) 5 weeks of treatment did not significantly affect the liver NAS of nSTZ+HFD mice. The experimental results are shown in Figure 9.

1.3天狼星紅染色圖片。 1.3 Picture of Sirius Red Staining.

天狼星紅染色結果顯示，nSTZ+HFD小鼠肝臟組織發生較明顯竇周、3區合併門脉周圍纖維化改變，另見個別樣本發生疑似橋接纖維化。己酮可可鹼(100mpk,po.,t.i.d.)與化合物1(30mpk,po.,b.i.d.)5周治療組均不同程度的減輕STZ+HFD小鼠上述肝組織區域內發生纖維化的程度，其中化合物1(30mpk,po.,b.i.d.)5周治療組較明顯改善肝臟組織發生竇周合併門脉周圍纖維化的形態學改變。實驗結果見圖10。 The results of Sirius red staining showed that the liver tissue of nSTZ+HFD mice had more obvious changes in perisinus, zone 3 and periportal fibrosis. In addition, some samples had suspected bridging fibrosis. Both the pentoxifylline (100mpk, po., tid) and compound 1 (30mpk, po., bid) treatment groups for 5 weeks reduced the degree of fibrosis in the liver tissue area of STZ+HFD mice to varying degrees. The compound 1 (30mpk, po., bid) The 5-week treatment group significantly improved the morphological changes of liver tissue with peri-sinus and periportal fibrosis. The experimental results are shown in Figure 10.

1.4纖維化評分。 1.4 Fibrosis score.

nSTZ+HFD小鼠肝臟纖維化評分顯著上升，己酮可可鹼(100mpk,po.,t.i.d.)5周治療有降低nSTZ+HFD小鼠肝臟纖維化趨勢，但經統計學分 析，沒有顯著性，而化合物1(30mpk,po.,b.i.d.)5周治療能夠明顯降低nSTZ+HFD小鼠肝臟纖維化評分。實驗結果見圖11。 The liver fibrosis score of nSTZ+HFD mice increased significantly. Pentoxifylline (100mpk, po., t.i.d.) treatment for 5 weeks reduced the tendency of liver fibrosis in nSTZ+HFD mice, but statistically According to the analysis, there is no significant, and compound 1 (30mpk, po., b.i.d.) 5 weeks treatment can significantly reduce the liver fibrosis score of nSTZ+HFD mice. The experimental results are shown in Figure 11.

2．肝臟生物標記物mRNA檢測結果。 2. Results of liver biomarker mRNA detection.

nSTZ+HFD小鼠肝臟TNF-α與TGF-β1 mRNA顯著上升，己酮可可鹼(100mpk,po.,t.i.d.)5周治療對nSTZ+HFD小鼠肝臟TNF-α與TGF-β1 mRNA水平沒有明顯性影響，化合物1(30mpk,po.,b.i.d.)給藥5周nSTZ+HFD小鼠肝臟TNF-α與TGF-β1mRNA水平顯著降低。nSTZ+HFD小鼠肝臟Collagen 1α1 mRNA有上升趨勢，化合物1(30mpk,po.,b.i.d.)給藥5周nSTZ+HFD小鼠肝臟Collagen mRNA有下降趨勢，但統計學分析沒有顯著性，需進一步研究分析。實驗結果見圖12。 TNF-α and TGF-β1 mRNA in the liver of nSTZ+HFD mice increased significantly. Pentoxifylline (100mpk, po., tid) for 5 weeks had no significant effect on the levels of TNF-α and TGF-β1 mRNA in the liver of nSTZ+HFD mice. Sexual effects, compound 1 (30mpk, po., bid) administration for 5 weeks nSTZ+HFD mice liver TNF-α and TGF-β1 mRNA levels were significantly reduced. Collagen 1α1 mRNA in the liver of nSTZ+HFD mice showed an upward trend. Compound 1 (30mpk, po., bid) was administered for 5 weeks. Collagen mRNA in the liver of nSTZ+HFD mice showed a downward trend. However, the statistical analysis was not significant, and further research is needed. analysis. The experimental results are shown in Figure 12.

3.血清生化指標檢測結果。 3. Serum biochemical index test results.

nSTZ+HFD小鼠血清ALT與TC水平顯著升高，己酮可可鹼(100mpk,po.,t.i.d.)與化合物1(30mpk,po.,b.i.d.)5周治療並不能顯著影響nSTZ+HFD小鼠血清ALT與TC水平。實驗結果見圖13。 The serum ALT and TC levels of nSTZ+HFD mice were significantly increased. Pentoxifylline (100mpk, po., tid) and compound 1 (30mpk, po., bid) for 5 weeks did not significantly affect the serum of nSTZ+HFD mice ALT and TC levels. The experimental results are shown in Figure 13.

總結。 to sum up.

1.新生鼠注射低劑量鏈脲黴素(streptozotocin,STZ)並進行高脂飲食飼養1周後形成糖尿病合併脂肪肝。持續的高脂飲食導致肝脂肪沉積，肝小葉炎症的增加，並伴隨泡沫樣巨噬細胞浸潤。nSTZ+HFD小鼠模型模擬人類糖尿病-非酒精性脂肪性肝炎-肝細胞性肝癌的病理進程，是廣泛使用的非酒精性脂肪性肝炎動物模型。nSTZ+HFD模型小鼠肝臟組織學HE染色圖片顯示出明顯的脂肪變性，氣球樣變與小葉炎症，天狼星紅染色顯示出明顯的纖維化，肝臟炎症生物標志物TNF-αmRNA顯著升高，纖維化生物標志物TGF-β1 mRNA顯著升高，血清ALT與TG水平較正常小鼠顯著升高。 1. Newborn rats were injected with low-dose streptozotocin (STZ) and fed with a high-fat diet for 1 week, and then developed diabetes and fatty liver. Continuous high-fat diet leads to liver fat deposition and increased inflammation of liver lobules, accompanied by foamy macrophage infiltration. The nSTZ+HFD mouse model simulates the pathological process of human diabetes-non-alcoholic steatohepatitis-hepatocellular carcinoma, and is a widely used animal model of non-alcoholic steatohepatitis. The histological HE staining pictures of the liver of nSTZ+HFD model mice showed obvious steatosis, ballooning and lobular inflammation, Sirius red staining showed obvious fibrosis, liver inflammation biomarker TNF-αmRNA was significantly increased, and fibrosis The biomarker TGF-β1 mRNA was significantly increased, and the serum ALT and TG levels were significantly higher than that of normal mice.

2.化合物1(30mpk,po.,b.i.d.)5周給藥後，nSTZ+HFD小鼠肝臟纖維化顯著改善，肝臟TNF-α與TGF-β1 mRNA水平顯著降低。 2. After 5 weeks of administration of compound 1 (30mpk, po., b.i.d.), the liver fibrosis of nSTZ+HFD mice was significantly improved, and the liver TNF-α and TGF-β1 mRNA levels were significantly reduced.

3.化合物1(30mpk,po.,b.i.d.)5周給藥，對nSTZ+HFD小鼠肝臟NAS，血清ALT，AST，TG與TC水平無顯著性影響。 3. Compound 1 (30mpk, po., b.i.d.) administered for 5 weeks has no significant effect on liver NAS, serum ALT, AST, TG and TC levels in nSTZ+HFD mice.

實驗例6：抗大鼠膽汁淤積型肝纖維化的藥效研究。 Experimental Example 6: Study on the pharmacodynamics of anti-cholestatic liver fibrosis in rats.

主要任務：評估化合物1對大鼠膽汁淤積型肝纖維化模型肝臟組織學，以及血清ALT，AST，TG和TC水平的影響。 Main task: To evaluate the effect of compound 1 on liver histology and serum ALT, AST, TG and TC levels in a rat cholestatic liver fibrosis model.

擬解决的問題：測試化合物1對大鼠膽汁淤積型肝纖維化模型肝臟組織學，血清ALT，AST，TG和TC水平的影響，評估化合物對大鼠膽汁淤積型肝纖維化模型肝功能保護，降低肝臟炎症和纖維化的作用。 The problem to be solved: To test the effect of compound 1 on liver histology, serum ALT, AST, TG and TC levels in a rat cholestatic liver fibrosis model, and evaluate the compound's protection of liver function in a rat cholestatic liver fibrosis model Reduce liver inflammation and fibrosis.

實驗模型：(ANIT)誘導的膽汁淤積型肝纖維化大鼠模型。 Experimental model: (ANIT)-induced cholestatic liver fibrosis rat model.

實驗動物：SD大鼠(雄性，上海斯萊克實驗動物有限責任公司) Experimental animals: SD rats (male, Shanghai Slack Laboratory Animal Co., Ltd.)

實驗試劑：ANIT(1-NAPHTHYL ISOTHIOCYANATE，Aldrich-N4525)；HPMC(Hydroxypropyl methyl cellulose，Sigma-H7509。 Experimental reagents: ANIT (1-NAPHTHYL ISOTHIOCYANATE, Aldrich-N4525); HPMC (Hydroxypropyl methyl cellulose, Sigma-H7509).

實驗方法。 experimental method.

1.實驗流程。 1. Experimental process.

Figure 105135042-A0305-02-0190-175
Figure 105135042-A0305-02-0190-175

2.體內實驗方法。 2. In vivo experimental methods.

訂購了90隻大鼠，動物到的當天，按照組與組之間差異最小的原則進行分組，每組12隻，每籠4隻。動物到達設施後，適應期為5天。適應 期內，給動物提供正常的飲食和飲水，每天監測動物的健康狀况。如發現任何異常或者感染情况，該大鼠則需要被剔除出試驗組。 90 rats were ordered. On the day the animals arrived, they were grouped according to the principle of the smallest difference between groups, with 12 rats in each group and 4 rats in each cage. After the animals arrive at the facility, the acclimatization period is 5 days. adapt During the period, the animals were provided with normal diet and drinking water, and the health of the animals was monitored every day. If any abnormality or infection is found, the rat needs to be removed from the experimental group.

第0天，先給藥。2小時後，將除第一組外的其他所有籠盒內的正常飼料換成ANIT添加料，添加量為大鼠正常三天的攝入量(這個量是根據之前的實驗結果得出)。ANIT添加料每三天更新一次。第一組的大鼠在整個實驗過程中食用正常飼料。 On day 0, the drug is administered first. After 2 hours, the normal feed in all cages except the first group was replaced with ANIT supplements, and the amount added was the normal three-day intake of the rats (this amount is based on the results of the previous experiment). ANIT additives are updated every three days. The rats in the first group ate normal feed throughout the experiment.

Figure 105135042-A0305-02-0191-176
Figure 105135042-A0305-02-0191-176

所有大鼠在第8天上午被撤去食物，直到實驗終點，禁食時間5小時以上。安樂死方法：戊巴比妥鈉麻醉後心臟採血處死。(收集盡可能多的血液)。 All rats were withdrawn from food in the morning of the 8th day until the end of the experiment, and the fasting time was more than 5 hours. Euthanasia method: blood was collected from the heart after pentobarbital sodium anesthesia. (Collect as much blood as possible).

1)收集血液2mL；收集血清750μL，分裝於3個離心管中。(血清收集方式：10000rpm，4℃離心10分鐘，收集血清，置於乾冰上轉移至-80℃保存)；2)收集動物肝臟，用PBS沖洗；3)用滅菌的紙巾拭乾，稱重並記錄；4)拍照整個離體肝臟； 5)從肝左葉最大直徑處，切取一條，寬約3mm，浸入10倍於組織體積的10%福爾馬林溶液(純水配置，需提前調PH值為中性)中；6)剩餘的肝左葉，取100mg的兩塊，剪成小塊分裝於兩個凍存管，用於肝臟TG/TC的檢測；7)肝臟中葉，一分為二，各取一部分放入兩個凍存管中。 1) Collect 2 mL of blood; collect 750 μL of serum and distribute them in 3 centrifuge tubes. (Serum collection method: 10000rpm, 4℃ centrifugation for 10 minutes, collect serum, place on dry ice and transfer to -80℃ for storage); 2) Collect animal liver, rinse with PBS; 3) Wipe dry with sterile paper towel, weigh and Record; 4) Take pictures of the entire isolated liver; 5) Cut one piece from the largest diameter of the left liver lobe, with a width of about 3 mm, and immerse it in 10 times the volume of the tissue in a 10% formalin solution (pure water configuration, pH needs to be adjusted in advance to be neutral); 6) the rest The left lobe of the liver, take two pieces of 100mg, cut into small pieces and put them into two cryopreservation tubes for the detection of TG/TC of the liver; 7) The middle lobe of the liver, one is divided into two, each part is taken into two Freeze in the tube.

3．組織學分析方法。 3. Histological analysis methods.

3.1組織處理。 3.1 Organizational processing.

進行肝臟組織脫水，石蠟塊製作，石蠟切片，HE染色石蠟片厚3μm，天狼星紅染色石蠟片厚4μm。遵循KCI病理標準染色SOP分別進行HE染色和天狼猩紅染色；HE染色的片子由兩位病理學專家進行打分(評判標準見病理分報告)，最終取二者的平均值分析各組的肝臟損傷程度；纖維化染色切片經Digital Pathscope 4S掃描儀全片掃描後，進行整片分析。 The liver tissue was dehydrated, paraffin blocks were made, and paraffin sections were performed. The HE stained paraffin slices were 3 μm thick, and the Sirius red stained paraffin slices were 4 μm thick. Follow the KCI pathology standard staining SOP for HE staining and Sirius scarlet staining; HE stained films were scored by two pathologists (see pathology score report for the criteria), and finally the average of the two was used to analyze the liver damage in each group Degree: After the stained fibrosis section is scanned by the Digital Pathscope 4S scanner, the whole section is analyzed.

3.2大鼠膽汁淤積模型炎症病理評分標準 3.2 Inflammatory pathology scoring criteria for rat cholestasis model

Figure 105135042-A0305-02-0192-177
Figure 105135042-A0305-02-0192-177
Figure 105135042-A0305-02-0193-178
Figure 105135042-A0305-02-0193-178

實驗結果。 Experimental results.

1.組織學結果。 1. Histological results.

1.1 HE染色圖片及結果。 1.1 HE staining pictures and results.

根據HE染色結果(圖14)，按照炎症病理評分標準對肝臟炎症水平進行打分(圖15)，對結果進行t檢驗。健康對照組顯著性低於模型組，但陽性對照INT-747給藥組的分值則顯著性升高。己酮可可鹼-100mg/kg和化合物1均未觀察到對肝臟炎症的改善作用。 According to the HE staining results (Figure 14), the liver inflammation level was scored according to the inflammation pathology scoring standard (Figure 15), and the results were subjected to t-test. The healthy control group was significantly lower than the model group, but the score of the positive control INT-747 administration group was significantly higher. Neither pentoxifylline -100 mg/kg nor compound 1 was observed to improve liver inflammation.

1.2天狼星紅染色圖片及纖維化評分。 1.2 Sirius red stained picture and fibrosis score.

肝組織切片通過天狼星紅染色(圖16)，經過Digital pathscope 4S全片掃描，對全景切片進行分析，計算膠原纖維在切片中的百分比，以此來判斷各組動物經過化合物處理後的纖維化程度變化(圖17)。結果顯示模型組的分值顯著性高於健康對照組，顯示肝纖維化造模成功。與模型組相比，化合物1在本研究的最高劑量組10mg/kg時，對肝臟的纖維化程度有抑制作用(p=0.03)，抑制率為17%，與INT-747對肝纖維化的改善程度相似(p=0.008)。而己酮可可鹼在100mg/kg劑量對肝纖維化沒有改善作用。 The liver tissue slices were stained with Sirius Red (Figure 16), and then scanned by Digital pathscope 4S. The panoramic slices were analyzed to calculate the percentage of collagen fibers in the slices to determine the degree of fibrosis in each group of animals after treatment with the compound. Changes (Figure 17). The results showed that the score of the model group was significantly higher than that of the healthy control group, indicating that the model of liver fibrosis was successful. Compared with the model group, compound 1 has an inhibitory effect on liver fibrosis at 10 mg/kg in the highest dose group in this study (p=0.03), with an inhibition rate of 17%, which is comparable to the effect of INT-747 on liver fibrosis. The degree of improvement was similar (p=0.008). However, pentoxifylline at a dose of 100 mg/kg has no effect on liver fibrosis.

2.血清生化指標檢測結果(表4)。 2. Serum biochemical index test results (Table 4).

相對於正常組，模型組的ALT、AST、ALP、TBA、TB、DB和IB均顯著性增高，說明造模成功。與模型組相比，INT-747組一定程度上可以降低血清中ALT和AST的水平，分別降低18%和25%(均值比較)，但是在本次試驗中並沒有統計學上的差異；己酮可可鹼和化合物1均沒有顯著降低AST和ALT的作用。所有給藥組都沒有降低血清中ALP的水平。 Compared with the normal group, the ALT, AST, ALP, TBA, TB, DB, and IB of the model group were significantly increased, indicating that the model was successful. Compared with the model group, the INT-747 group can reduce the levels of ALT and AST in the serum to a certain extent, by 18% and 25% respectively (compared to the average value), but there is no statistical difference in this test; Neither Ketoxifylline nor Compound 1 significantly reduced AST and ALT. All the administration groups did not reduce the level of ALP in serum.

總膽汁酸(TBA)水平，模型組與正常組相比顯著性增高，升高約9倍。與模型組相比，INT-747，20mg/kg處理組的TBA水平顯著性降低， 降低約75%。化合物1處理組有一定的降低總膽汁酸的作用，降低分別為37%、26%、33%，但是不具有統計學上的意義。 The total bile acid (TBA) level in the model group was significantly higher than that in the normal group, which was about 9 times higher. Compared with the model group, the TBA level of the INT-747, 20mg/kg treatment group was significantly reduced, Reduce by about 75%. The compound 1 treatment group had a certain effect of reducing total bile acids, which were reduced by 37%, 26%, and 33%, but they were not statistically significant.

TB、DB、IB的檢測結果顯示，與正常對照組相比，模型組的數值顯著性升高，分別升高53倍、62倍、36倍；而將藥物處理組與模型組相比，陽性對照組INT-747具有顯著性的降低TB、DB、IB的作用，分別降低53%、54%、49%；化合物1在高劑量作用下，TB、DB、IB的水平有所下降，且可以看出相應的劑量依賴性，但與模型組相比不具有統計學意義實驗結果。 The test results of TB, DB, and IB showed that compared with the normal control group, the value of the model group was significantly increased by 53 times, 62 times, and 36 times, respectively; while comparing the drug treatment group with the model group, the value was positive The control group INT-747 has a significant effect of reducing TB, DB, and IB by 53%, 54%, and 49%, respectively. Compound 1 has reduced the levels of TB, DB, and IB at high doses. See the corresponding dose dependence, but the experimental results are not statistically significant compared with the model group.

Figure 105135042-A0305-02-0195-179
Figure 105135042-A0305-02-0195-179

總結： to sum up:

1)在ANIT誘導的膽汁淤積型肝纖維化大鼠模型中，模型組動物血清中的ALT/AST/ALP在血清中的數值顯著性高於健康對照組；TBA/TB/DB/IB在血清中的檢測結果也顯著性升高；炎症評分結果中，與健康對照組相比，模型組的纖維化程度明顯升高；病理纖維化結果中，與健康對照組相比，模型組的纖維化程度明顯升高。 1) In the rat model of cholestasis-induced liver fibrosis induced by ANIT, the ALT/AST/ALP in the serum of the model group was significantly higher than that in the healthy control group; TBA/TB/DB/IB in the serum In the results of the inflammation score, compared with the healthy control group, the degree of fibrosis in the model group was significantly increased; in the results of pathological fibrosis, compared with the healthy control group, the fibrosis in the model group The degree is significantly increased.

2)化合物1(10mpk,po.,b.i.d.)處理組在病理纖維化程度上表現了明顯的抗纖維化作用。 2) Compound 1 (10mpk, po., b.i.d.) treatment group showed significant anti-fibrosis effect in the degree of pathological fibrosis.

3)化合物1各處理組，對血清ALT，AST，TG與TC水平無顯著性影響。 3) Each treatment group of Compound 1 has no significant effect on serum ALT, AST, TG and TC levels.

實驗例7：四氯化碳誘導的小鼠肝纖維化模型藥效研究。 Experimental Example 7: Study on the efficacy of a mouse liver fibrosis model induced by carbon tetrachloride.

主要任務：評估化合物1對四氯化碳誘導的小鼠肝纖維化模型肝臟組織學，以及血清ALT，AST，TG和TC水平的影響。 Main task: To evaluate the effect of compound 1 on liver histology and serum ALT, AST, TG and TC levels in a mouse liver fibrosis model induced by carbon tetrachloride.

擬解决的問題：測試化合物1對四氯化碳誘導的小鼠肝纖維化模型肝臟組織學，血清ALT，AST，TG和TC水平的影響，評估化合物對四氯化碳誘導的小鼠肝纖維化模型肝功能保護，降低肝臟損傷和纖維化的作用。 The problem to be solved: To test the effect of compound 1 on liver histology, serum ALT, AST, TG and TC levels in a mouse liver fibrosis model induced by carbon tetrachloride, and to evaluate the compound's effect on carbon tetrachloride-induced liver fibrosis in mice The chemical model protects liver function and reduces liver damage and fibrosis.

實驗模型：四氯化碳誘導的小鼠肝纖維化模型。 Experimental model: a mouse liver fibrosis model induced by carbon tetrachloride.

實驗動物：C57BL/6小鼠(雄性，上海靈暢生物科技有限責任公司) Experimental animals: C57BL/6 mice (male, Shanghai Lingchang Biotechnology Co., Ltd.)

實驗試劑： Experimental reagents:

1.CCl4(江蘇強盛功能化學股份有限公司) 1. CCl 4 (Jiangsu Qiangsheng Functional Chemical Co., Ltd.)

2.橄欖油(阿拉丁工業公司，0108686-500ml) 2. Olive oil (Aladdin Industries, 0086686-500ml)

3.HPMC(Hydroxypropyl methyl cellulose，Sigma-H7509) 3. HPMC (Hydroxypropyl methyl cellulose, Sigma-H7509)

實驗方法。 experimental method.

1.實驗流程。 1. Experimental process.

Figure 105135042-A0305-02-0197-182
Figure 105135042-A0305-02-0197-182

2.體內實驗方法。 2. In vivo experimental methods.

訂購了108隻小鼠，其中3隻備用。動物到的當天，按照組與組之間差異最小的原則進行分組，每組15隻。動物到達設施後，適應期為5天。適應期內，每天監測動物的健康狀况。如發現任何異常或者感染情况，該小鼠則需要被剔除出試驗組。25% CCl4溶液用純橄欖油配製，現配現用。除第一組的動物外，注射時間為第0天、第4天、第8天、第12天，腹腔注射其他所有組的小鼠，根據體重給藥，2mL/kg。第一組的小鼠，腹腔注射純橄欖油。 108 mice were ordered and 3 of them were spared. On the day the animals arrive, they are grouped according to the principle of the smallest difference between groups, with 15 animals in each group. After the animals arrive at the facility, the acclimatization period is 5 days. During the adaptation period, the health of the animals was monitored daily. If any abnormality or infection is found, the mouse needs to be removed from the experimental group. The 25% CCl 4 solution is prepared with pure olive oil, and it is prepared for immediate use. Except for the animals in the first group, the injection time was on day 0, day 4, day 8, and day 12. The mice of all other groups were injected intraperitoneally with 2 mL/kg according to body weight. The mice in the first group were injected with pure olive oil intraperitoneally.

Figure 105135042-A0305-02-0197-181
Figure 105135042-A0305-02-0197-181

所有小鼠在第14天上午被撤去食物，直到實驗終點，禁食時間5小時以上。安樂死方法：麻醉後眼眶後靜脈叢採血後，脫頸確認死亡。(收集盡可能多的血液)。 All mice were withdrawn food on the morning of the 14th day until the end of the experiment, and the fasting time was more than 5 hours. Euthanasia method: After anesthesia, blood was collected from the retroorbital venous plexus, and the neck was taken off to confirm death. (Collect as much blood as possible).

1)收集盡可能多的血液；操作收集血清250ul，分裝於相應離心管中。(血清收集方式：10000rpm，4度離心10分鐘，收集血清，置於乾冰 上轉移至-80度保存。) 1) Collect as much blood as possible; operate to collect 250ul of serum and distribute it in corresponding centrifuge tubes. (Serum collection method: 10000rpm, 4 degrees centrifugation for 10 minutes, collect the serum, place on dry ice Transfer to -80 degrees to save. )

2)收集動物肝臟，用PBS沖洗 2) Collect animal liver and rinse with PBS

3)用滅菌的紙巾拭乾，稱重並記錄； 3) Wipe dry with sterilized paper towels, weigh and record;

4)拍照； 4) Take pictures;

5)從肝左葉最大直徑處，切取一條，寬約3mm，浸入10倍體積的10%福爾馬林溶液(純水配置，需提前調PH值為中性)中，垂直放置固定； 5) Cut one piece from the largest diameter of the left lobe of the liver, with a width of about 3mm, immerse it in 10 times the volume of 10% formalin solution (pure water configuration, need to adjust the pH value in advance to neutral), and place it vertically and fix it;

6)剩餘的肝左葉，剪成小塊分裝於兩個凍存管，用於檢測肝臟TG/TC； 6) The remaining left lobe of the liver is cut into small pieces and placed in two cryopreservation tubes for the detection of liver TG/TC;

7)肝臟中葉，一分為二，放入兩個凍存管中，作為蛋白分析和mRNA檢測的樣品。 7) The middle lobe of the liver is divided into two and placed in two cryopreservation tubes as samples for protein analysis and mRNA detection.

需要注意：第6)和7)的樣品收集後需迅速浸入液氮中，且需要保證凍存管在扔入液氮後完全浸入液氮中。 Note: The samples in Sections 6) and 7) should be quickly immersed in liquid nitrogen after being collected, and the cryotube should be completely immersed in liquid nitrogen after being thrown into liquid nitrogen.

3．組織學分析方法。 3. Histological analysis methods.

3.1組織處理。 3.1 Organizational processing.

進行肝臟組織脫水，石蠟塊製作，石蠟切片，HE染色石蠟片厚3μm，天狼星紅染色石蠟片厚4μm。遵循KCI病理標準染色SOP分別進行HE染色和天狼猩紅染色。HE染色的片子由兩位病理學專家進行打分，最終取二者的平均值分析各組的肝臟損傷程度；纖維化染色切片。經Digital Pathscope 4S掃描儀全片掃描後，進行整片分析。 The liver tissue was dehydrated, paraffin blocks were made, and paraffin sections were performed. The HE stained paraffin slices were 3 μm thick, and the Sirius red stained paraffin slices were 4 μm thick. Follow the KCI pathological standard staining SOP for HE staining and Sirius scarlet staining respectively. The HE-stained slides were scored by two pathologists, and finally the average of the two was used to analyze the degree of liver damage in each group; fibrosis stained sections. After scanning the whole slice by the Digital Pathscope 4S scanner, the whole slice is analyzed.

3.2四氯化碳誘導肝損傷動物模型病理評分標準。 3.2 The pathological scoring criteria for animal models of liver injury induced by carbon tetrachloride.

Figure 105135042-A0305-02-0199-183
Figure 105135042-A0305-02-0199-183

實驗結果。 Experimental results.

1.組織學結果。 1. Histological results.

1.1 HE染色圖片及結果。 1.1 HE staining pictures and results.

肝臟病理切片經過HE染色後(圖18)，按照病理評分標準對炎症水平、氣球樣變、水樣變性和壞死四項指標分別打分。結果顯示，與健康對照組相比，模型組四個指標的分值均極顯著性升高，證明造模成功(圖19，圖20，圖21，圖22)。 After the liver pathological section was stained with HE (Figure 18), the four indicators of inflammation, ballooning, watery degeneration, and necrosis were scored according to the pathological scoring criteria. The results showed that compared with the healthy control group, the scores of the four indicators of the model group were all extremely significantly increased, proving that the model was successful (Figure 19, Figure 20, Figure 21, Figure 22).

與模型組相比，化合物1三個劑量組水樣變性和壞死的分值均較模型組降低(均有統計學意義)，且作用表現出了一定的劑量依賴性。且化合物1-10mg/kg組對水樣變性和壞死的改善作用與己酮可可鹼-100mg/kg組相當。相比模型組，化合物1-10mg/kg水樣變性和壞死評分分別降低0.93分和0.86分，己酮可可鹼-100mg/kg組水樣變性和壞死評分分別下降0.90和0.67分。 Compared with the model group, the scores of watery degeneration and necrosis in the three dose groups of compound 1 were lower than those of the model group (all statistically significant), and the effects showed a certain dose-dependence. And the improvement effect of compound 1-10mg/kg group on watery degeneration and necrosis is equivalent to that of pentoxifylline-100mg/kg group. Compared with the model group, the watery degeneration and necrosis scores of the compound 1-10 mg/kg decreased by 0.93 points and 0.86 points, respectively, and the watery degeneration and necrosis scores of the pentoxifylline-100 mg/kg group decreased by 0.90 and 0.67 points, respectively.

四個指標的評分進行加和的結果顯示，各個測試化合物對加和分值均有顯著改善。化合物1三個測試劑量中對肝臟損傷的均有顯著改善，有一定的劑量相關性，作用與己酮可可鹼-100mg/kg相當(圖23)。 The results of adding the scores of the four indicators show that each test compound has a significant improvement in the addition score. Compound 1 has a significant improvement in liver damage at the three tested doses, with a certain dose correlation, and the effect is equivalent to pentoxifylline-100mg/kg (Figure 23).

1.2天狼星紅染色圖片及纖維化評分。 1.2 Sirius red stained picture and fibrosis score.

天狼星紅染色結果(圖24)讀取纖維化面積(圖25)，模型組的分值顯著性高於健康對照組，顯示肝纖維化造模成功。與模型組相比，化合物1的高中低三個劑量組、己酮可可鹼和INT-747均可顯著降低肝臟組織的纖維化。 The results of Sirius Red staining (Figure 24) read the area of fibrosis (Figure 25). The score of the model group was significantly higher than that of the healthy control group, indicating that the model of liver fibrosis was successful. Compared with the model group, the three high, medium and low dose groups of compound 1, pentoxifylline and INT-747 can significantly reduce liver fibrosis.

2.血清生化指標檢測結果(表5)。 2. Serum biochemical index test results (Table 5).

相對於正常組，模型組的ALT、AST、TG、TC都顯著性增高，說明造模成功。 Compared with the normal group, the ALT, AST, TG, and TC of the model group were significantly higher, indicating that the model was successful.

與模型組相比，化合物1具有一定的降低ALT和AST的作用，且具有劑量依賴性，化合物1在1，3和10mg/kg的劑量分別使ALT降低24%，52%和56%，使AST分別降低23%，42%，52%。但只有化合物1-10mg/kg劑量，AST的改變具有統計學意義(p=0.02)。INT-747可顯著降低血清中ALT和AST的水平，分別降低75%和64%，另外INT-747也可使TG和TC有所下降，分別降低11%和12%。而將其他藥物處理組與模型組相比，但己酮可可鹼對ALT和AST、TG、TC均無改善作用。 Compared with the model group, compound 1 has a certain effect of reducing ALT and AST, and is dose-dependent. Compound 1 at the doses of 1, 3 and 10 mg/kg can reduce ALT by 24%, 52% and 56%, respectively. AST decreased by 23%, 42%, and 52%, respectively. But only with compound 1-10mg/kg dose, the change of AST was statistically significant (p=0.02). INT-747 can significantly reduce serum ALT and AST levels by 75% and 64%, respectively. In addition, INT-747 can also reduce TG and TC by 11% and 12%, respectively. Compared with the model group in other drug treatment groups, pentoxifylline had no effect on ALT, AST, TG, and TC.

Figure 105135042-A0305-02-0201-184
Figure 105135042-A0305-02-0201-184

總結： to sum up:

1)三個劑量的化合物1一定程度上都可以降低血清中的ALT/AST，且表現了一定的劑量依賴性，尤其最高級量10mg/kg可以顯著性減低AST；2)從HE染色結果看，化合物1可以顯著性改善肝臟的損傷，包括炎症水平、水樣變性程度、壞死程度，劑量10mg/kg處理組對這些指標的改善作用在三個劑量中最強；3)化合物1在三個測試劑量下，可以顯著性降低肝臟纖維化程度。 1) The three doses of Compound 1 can reduce the ALT/AST in the serum to a certain extent, and show a certain dose-dependent, especially the highest dose of 10mg/kg can significantly reduce AST; 2) From the results of HE staining Compound 1 can significantly improve liver damage, including inflammation level, degree of watery degeneration, and degree of necrosis. The 10 mg/kg treatment group has the strongest improvement effect on these indicators among the three doses; 3) Compound 1 was tested in three tests Under the dose, it can significantly reduce the degree of liver fibrosis.

Figure 105135042-A0304-11-0002-2
Figure 105135042-A0304-11-0002-2

Claims (10)

一種式(I)所示化合物、其互變異構體或其藥學上可接受的鹽在製備治療或預防肝病藥物中的應用，
Figure 105135042-A0305-02-0203-185
 其中， 或將結構單元
Figure 105135042-A0305-02-0203-196
替換為
Figure 105135042-A0305-02-0203-198
； L11選自空、C(R)(R’)；R、R’分別獨立地選自H、鹵素、OH、NH2、CN、任選被取代的1~6元烷基；任選地，R、R’可以環化成3-6元環烷基； A選自任選被取代的：環丙基、
Figure 105135042-A0305-02-0203-199
、
Figure 105135042-A0305-02-0203-200
、環戊基、環己基、 環氧戊基、苯基、吡啶基、吡嗪基、噁唑基、異噁唑基、噻唑基、雙環[1.1.1]戊烷； L12選自亞甲基、
Figure 105135042-A0305-02-0203-201
、
Figure 105135042-A0305-02-0203-202
、
Figure 105135042-A0305-02-0203-203
、
Figure 105135042-A0305-02-0203-205
、
Figure 105135042-A0305-02-0203-206
 R1選自任選被取代的1~6元烷基、3-6元環烷基或3-6元雜烷基；以及 R、R’、A、R1中取代基分別獨立地選自鹵素、OH、NH2、CN、CF3、
Figure 105135042-A0305-02-0204-223
、1~6元烷基、3-6元環烷基或3-6元雜烷基，每個基團取代基的 數目分別獨立地選自1、2或3；“雜”代表N、O、S、C(=O)、S(=O)、S(=O)2，每個基團上雜原子的數目選自1、2、3或4。 The use of a compound represented by formula (I), its tautomer or a pharmaceutically acceptable salt thereof in the preparation of a medicine for treating or preventing liver disease,
Figure 105135042-A0305-02-0203-185
 Among them, or the structural unit
Figure 105135042-A0305-02-0203-196
Replace with
Figure 105135042-A0305-02-0203-198
; L 11 is selected from empty, C(R)(R'); R and R'are independently selected from H, halogen, OH, NH 2 , CN, and optionally substituted 1- to 6-membered alkyl; optionally Ground, R and R'can be cyclized into 3-6 membered cycloalkyl; A is selected from optionally substituted: cyclopropyl,
Figure 105135042-A0305-02-0203-199
,
Figure 105135042-A0305-02-0203-200
, Cyclopentyl, cyclohexyl, epoxypentyl, phenyl, pyridyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, bicyclo[1.1.1]pentane; L 12 is selected from methylene base,
Figure 105135042-A0305-02-0203-201
,
Figure 105135042-A0305-02-0203-202
,
Figure 105135042-A0305-02-0203-203
,
Figure 105135042-A0305-02-0203-205
,
Figure 105135042-A0305-02-0203-206
 R 1 is selected from optionally substituted 1~6 membered alkyl, 3-6 membered cycloalkyl or 3-6 membered heteroalkyl; and the substituents in R, R', A, and R 1 are each independently selected from Halogen, OH, NH 2 , CN, CF 3 ,
Figure 105135042-A0305-02-0204-223
, 1~6 membered alkyl group, 3-6 membered cycloalkyl group or 3-6 membered heteroalkyl group, the number of substituents in each group is independently selected from 1, 2 or 3; "hetero" represents N, O , S, C(=O), S(=O), S(=O) 2 , the number of heteroatoms on each group is selected from 1, 2, 3, or 4. 如請求項1所述的應用，其中所述疾病選自非酒精性脂肪性肝炎和肝纖維化。 The use according to claim 1, wherein the disease is selected from non-alcoholic steatohepatitis and liver fibrosis. 如請求項1所述的應用，其中R、R’、A、R1中取代基分別獨立地選 自鹵素、CF3、CN、OH、Me、Et、正丙基、異丙基、環丙基、
Figure 105135042-A0305-02-0204-222
、
Figure 105135042-A0305-02-0204-208
 The application according to claim 1, wherein the substituents in R, R', A, and R 1 are independently selected from halogen, CF 3 , CN, OH, Me, Et, n-propyl, isopropyl, and cyclopropyl base,
Figure 105135042-A0305-02-0204-222
,
Figure 105135042-A0305-02-0204-208
 如請求項1-3任一項所述的應用，其中R、R’分別獨立地選自H、Me、CF3、Et。 The application according to any one of claims 1-3, wherein R and R'are independently selected from H, Me, CF 3 , and Et. 如請求項4所述的應用，其中，L11選自
Figure 105135042-A0305-02-0204-220
、
Figure 105135042-A0305-02-0204-218
、
Figure 105135042-A0305-02-0204-217
、
Figure 105135042-A0305-02-0204-221
、
Figure 105135042-A0305-02-0204-207
 The application according to claim 4, wherein L 11 is selected from
Figure 105135042-A0305-02-0204-220
,
Figure 105135042-A0305-02-0204-218
,
Figure 105135042-A0305-02-0204-217
,
Figure 105135042-A0305-02-0204-221
,
Figure 105135042-A0305-02-0204-207
 如請求項1-3任一項所述的應用，其中 A選自任選被取代的：
Figure 105135042-A0305-02-0204-210
、
Figure 105135042-A0305-02-0204-211
、
Figure 105135042-A0305-02-0204-213
、
Figure 105135042-A0305-02-0204-214
、
Figure 105135042-A0305-02-0204-215
、
Figure 105135042-A0305-02-0204-186
 The application according to any one of claims 1-3, wherein A is selected from optionally substituted:
Figure 105135042-A0305-02-0204-210
,
Figure 105135042-A0305-02-0204-211
,
Figure 105135042-A0305-02-0204-213
,
Figure 105135042-A0305-02-0204-214
,
Figure 105135042-A0305-02-0204-215
,
Figure 105135042-A0305-02-0204-186
 如請求項6所述的應用，其中A選自
Figure 105135042-A0305-02-0205-225
、
Figure 105135042-A0305-02-0205-226
、
Figure 105135042-A0305-02-0205-227
、
Figure 105135042-A0305-02-0205-187
 The application according to claim 6, wherein A is selected from
Figure 105135042-A0305-02-0205-225
,
Figure 105135042-A0305-02-0205-226
,
Figure 105135042-A0305-02-0205-227
,
Figure 105135042-A0305-02-0205-187
 如請求項1-3任一項所述的應用，其中R1選自Me、CHF2、CF3、Et、 CH2CF3、異丙基、
Figure 105135042-A0305-02-0205-228
、環丙基、
Figure 105135042-A0305-02-0205-230
、
Figure 105135042-A0305-02-0205-232
、
Figure 105135042-A0305-02-0205-234
、
Figure 105135042-A0305-02-0205-189
 The application according to any one of claims 1-3, wherein R 1 is selected from Me, CHF 2 , CF 3 , Et, CH 2 CF 3 , isopropyl,
Figure 105135042-A0305-02-0205-228
, Cyclopropyl,
Figure 105135042-A0305-02-0205-230
,
Figure 105135042-A0305-02-0205-232
,
Figure 105135042-A0305-02-0205-234
,
Figure 105135042-A0305-02-0205-189
 一種下式化合物在製備治療或預防肝病藥物中的應用，
Figure 105135042-A0305-02-0205-191
Figure 105135042-A0305-02-0206-193
Figure 105135042-A0305-02-0207-194
 An application of a compound of the following formula in the preparation of a medicine for treating or preventing liver disease,
Figure 105135042-A0305-02-0205-191
Figure 105135042-A0305-02-0206-193
Figure 105135042-A0305-02-0207-194
 一種下式化合物在製備治療或預防肝病藥物中的應用，
Figure 105135042-A0305-02-0207-195
 An application of a compound of the following formula in the preparation of a medicine for treating or preventing liver disease,
Figure 105135042-A0305-02-0207-195
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