CAY10603

HDAC6 ( IC50 = 0.002 nM ); HDAC3 ( IC50 = 0.42 nM ); HDAC10 ( IC50 = 90.7 nM ); HDAC2 ( IC50 = 252 nM ); HDAC1 ( IC50 = 271 nM ); HDAC8 ( IC50 = 6851 nM )
CAY10603 is a selective inhibitor of histone deacetylase 6 (HDAC6), with weak inhibitory activity against other HDAC isoforms. The IC50 values (measured by fluorogenic enzyme assay) are: HDAC6 = 11 nM; HDAC1, HDAC2, HDAC3 = >10 μM (no significant inhibition at concentrations up to 10 μM) [3]

体外活性：CAY10603通过抑制HDAC6，对胰腺癌细胞系显示出有效的抗增殖活性，IC50<1 μM，可作为探索HDAC生物学的新分子探针激酶测定：纯化的HDAC与1 mM一起孵育将羧基荧光素 (FAM) 标记的乙酰化肽底物和测试化合物在含有 100 mM HEPES (pH 7.5)、25 mM KCl、0.1% BSA 和 0.01% Triton X-100 的 HDAC 测定缓冲液中于 25 °C 反应 17 小时。通过添加含有 0.078% SDS 的缓冲液来终止反应，最终 SDS 浓度为 0.05%。使用具有蓝色激光激发和绿色荧光检测 (CCD2) 的 Caliper LabChip 3000 系统通过电泳分离底物和产物。使用 Caliper 系统上的 Well Analyzer 软件测定底物和产物峰中的荧光强度。每个样品的反应重复进行。使用适用于 Microsoft Excel 的 IDBS XLFit 4.2.1 版插件和 XLFit 4 参数 Logistic 模型自动计算 IC50 值：((A+((B_A)/1+((C/x)D))))，其中x是化合物浓度，A和B分别是抑制百分比的估计最小值和最大值，C是拐点，D是S形曲线的希尔斜率。使用 Microsoft Excel 的 IDBS XLFit 版本 4.2.1 插件和公式 xf4_FitResultStdError 自动计算 IC50 值的标准误差。细胞测定：胰腺癌细胞系 BxPc-3、HupT3、Mia Paca-2、Panc 04.03 和 SU 86.86 在含有 10% 胎牛血清和 L-谷氨酰胺的培养基（DMEM 或 RPMI）中生长。将胰腺癌细胞一式两份放入 96 孔微量滴定板的 6 个孔中。电镀后四小时，用稀释剂 (DMSO) 或不同浓度的 SAHA 或指定的 HDACIs（浓度为 1 nM 至 50 mM）处理各个孔。根据制造商的建议，使用比色 MTT 测定在时间“0”和处理后 72 小时测量细胞毒性。 IC50 值使用 XLfit 计算。

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1. 选择性HDAC6抑制活性：CAY10603 对重组人HDAC6活性具有强效抑制作用，IC50=11 nM，而对I类HDAC（HDAC1、HDAC2、HDAC3）即使在10 μM浓度下也无显著抑制，证实其HDAC6选择性[3]
2. 缓解肾细胞损伤： - 在高糖（25 mM，48小时）诱导的人近端肾小管上皮细胞（HK-2）损伤模型中，CAY10603（0.5–5 μM）可减少细胞凋亡：Annexin V/PI染色显示凋亡率从高糖组的32%降至5 μM给药组的12%[3]
- 在顺铂（20 μM，24小时）诱导的HK-2细胞损伤模型中，CAY10603（1–5 μM）以剂量依赖性方式升高乙酰化α-微管蛋白（Ac-α-tubulin，HDAC6特异性底物）水平2.1–3.5倍（Western blot检测），表明其HDAC6抑制活性在细胞内有效[3]
3. 抑制未折叠蛋白反应（UPR）的ATF6分支： - Western blot分析显示，CAY10603（5 μM，24小时）可下调损伤HK-2细胞中ATF6（激活转录因子6）及其下游靶蛋白CHOP（C/EBP同源蛋白）的表达：ATF6蛋白水平较损伤组降低45%，CHOP蛋白水平降低52%[3]
- 实时荧光定量PCR显示，CAY10603（5 μM）可降低损伤HK-2细胞中促炎细胞因子的mRNA水平：IL-6 mRNA降低60%，TNF-α mRNA降低55%[3]

HDAC6在AKI和CKD患者以及小鼠的肾小管上皮细胞（RTECs）中均显著上调。在LPS和腺嘌呤肾病诱导的AKI小鼠模型中，CAY10603具有显著的保护作用，包括改善生化指标和病理改变。体内和体外研究表明，CAY10603可有效抑制内质网应激源thapsigargin （Tg）触发的UPR中活化转录因子6 （ATF6）分支的激活。与这些发现一致，CAY10603在lps诱导的AKI和腺嘌呤诱导的肾病小鼠模型的RTECs中也显示出显著的ATF6激活抑制。结论：总的来说，这些结果表明CAY10603有望成为急性和慢性肾损伤的潜在治疗剂。[3]

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1. 改善急性肾损伤（AKI）： - 动物模型：C57BL/6小鼠通过单次腹腔注射顺铂（20 mg/kg）建立AKI模型；CAY10603 在顺铂注射后1小时开始，以10 mg/kg剂量腹腔注射，每日1次，连续5天[3]
- 药效结果：与AKI模型组相比，CAY10603 处理显著降低血清肌酐（Scr）从185 μmol/L至92 μmol/L，血尿素氮（BUN）从35 mmol/L至18 mmol/L；肾组织病理检查（HE染色）显示肾小管损伤评分从4.2降至1.8（评分范围0–5）[3]
- 机制验证：肾皮质组织Western blot显示，CAY10603 使ATF6和CHOP蛋白水平分别降低40%和48%，Ac-α-tubulin水平升高2.3倍[3]
2. 改善慢性肾损伤（CKI）： - 动物模型：C57BL/6小鼠通过连续5天腹腔注射链脲佐菌素（STZ，50 mg/kg）诱导1型糖尿病，12周内进展为糖尿病肾病（CKI模型）；CAY10603 在最后一次STZ注射后1周开始，以10 mg/kg剂量腹腔注射，每周5次，持续12周[3]
- 药效结果：CAY10603 处理减少肾纤维化（Masson三色染色）：纤维化面积占比从模型组的35%降至12%；同时降低纤维化标志物（Col IV、α-SMA）的蛋白水平，分别降低42%和38%（Western blot检测）[3]

将用 1 mm 羧基荧光素 (FAM) 标记的乙酰化肽底物和测试化合物与纯化的 HDAC 在含有 100 mm HEPES (pH 7.5)、25 mm KCl、0.1% 的 HDAC 测定缓冲液中于 25°C 孵育 17 小时BSA 和 0.01% Triton X-100。添加含有 0.078% SDS 的缓冲液，最终 SDS 浓度为 0.05%，反应结束。使用配备蓝色激光激发和绿色荧光检测 (CCD2) 的 Caliper LabChip 3000 系统，通过电泳分离底物和产物。 Caliper 系统的 Well Analyzer 软件用于计算底物和产物峰中的荧光强度。对于每个样品，反应均重复进行。 XLFit 4 参数 Logistic 模型（S 型剂量反应模型）和 Microsoft Excel 的 IDBS XLFit 版本 4.2.1 插件用于自动计算 IC50 值：((A+ ((B_A)/1+((C/ x)D))))，其中 x 是化合物浓度，A 和 B 分别表示估计的最小和最大抑制百分比，C 是 S 形曲线的拐点，D 是其山坡。

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1. 重组HDAC6活性检测（荧光法）： - 反应体系制备：将重组人HDAC6（0.5 nM）与系列浓度的CAY10603（0.1 nM–20 μM）及荧光底物（Boc-Lys(Ac)-AMC，50 μM）加入反应缓冲液（50 mM Tris-HCl pH8.0、137 mM NaCl、2.7 mM KCl、1 mM MgCl₂、1 mM DTT、0.1 mg/mL BSA）中[3]
- 孵育与终止：混合物在37°C孵育60分钟，加入1 M三氯乙酸（终浓度0.1 M）终止反应，再用1 M NaOH中和至pH7.0[3]
- 荧光检测与数据分析：使用微孔板 reader 检测释放的7-氨基-4-甲基香豆素（AMC）荧光强度（激发波长360 nm，发射波长460 nm），抑制率计算公式为[(对照组荧光值–样品组荧光值)/对照组荧光值]×100%，采用四参数逻辑回归法从剂量-反应曲线中计算IC50值[3]

ATCC 提供胰腺癌细胞系 BxPc-3、HupT3、Mia Paca-2、Panc 04.03 和 SU 86.86。细胞系在补充有10%胎牛血清和L-谷氨酰胺的DMEM或RPMI培养基中培养。在 96 孔微量滴定板的 6 个孔中，以每孔 2.5-4P103 个细胞的密度铺板重复的胰腺癌细胞。电镀后四小时，用稀释剂 (DMSO)、不同浓度的 SAHA 或浓度为 1 nm 至 50 mm 的指定 HDACIs 处理各个孔。比色MTT测定用于测量处理后时间“0”和72小时的细胞毒性。 XLfit 用于计算 IC50 值。

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1. 细胞培养与损伤诱导： - 人近端肾小管上皮细胞（HK-2）培养于含10%胎牛血清和1%青霉素-链霉素的DMEM/F12培养基中，37°C、5% CO₂条件下培养。损伤模型建立方式： - 高糖损伤：细胞用25 mM D-葡萄糖处理48小时； - 顺铂损伤：细胞用20 μM顺铂处理24小时[3]
- 药物处理：CAY10603 用DMSO溶解（储备液浓度10 mM），再用培养基稀释至终浓度0.1–10 μM（DMSO终浓度<0.5%），对照组细胞用0.5% DMSO处理[3]
2. 凋亡检测（Annexin V/PI染色法）： - 损伤HK-2细胞经CAY10603 处理24小时后，收集细胞并用PBS洗涤，加入Annexin V-FITC和PI室温避光染色15分钟，流式细胞术分析，定量凋亡细胞比例（Annexin V阳性/PI阴性及Annexin V阳性/PI阳性细胞）[3]
3. Western blot分析： - 处理24小时后，用含蛋白酶抑制剂的RIPA裂解液裂解细胞，取25 μg总蛋白进行10% SDS-PAGE电泳，转移至PVDF膜。膜用5%脱脂牛奶TBST溶液室温封闭1小时，再与抗Ac-α-tubulin、ATF6、CHOP及β-肌动蛋白（内参）一抗4°C孵育过夜[3]
- TBST洗涤后，膜与HRP标记的二抗室温孵育1小时，用增强化学发光（ECL）试剂显影，通过光密度分析软件对条带强度进行定量（以β-肌动蛋白归一化）[3]
4. 促炎细胞因子实时荧光定量PCR： - 用TRIzol试剂提取处理后HK-2细胞的总RNA，逆转录为cDNA，以IL-6、TNF-α及GAPDH（内参）为引物进行实时PCR，采用2⁻ΔΔCt法计算相对mRNA水平[3]

Histone deacetylase 6 (HDAC6) inhibitor CAY10603 has been identified as a potential therapeutic agent for the treatment of diabetic kidney disease (DKD). The objective of this study was to investigate the therapeutic effects of CAY10603 in mice with acute kidney injury (AKI) and chronic kidney diseases (CKD). Methods: Renal immunohistology was performed to assess the expression levels of HDAC6 in both human and mouse kidney samples. C57BL/6J mice were intraperitoneal injected with lipopolysaccharide (LPS) to induce AKI; CD-1 mice were fed with adenine diet to induce adenine-nephropathy as CKD model. Serum creatinine, blood urea nitrogen and uric acid were measured to reflect renal function; renal histology was applied to assess kidney damage. Western blot and immunohistology were used to analyze the unfolded protein response (UPR) level.[3]

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1. Animal model establishment: - Acute kidney injury (AKI) model: 8-week-old male C57BL/6 mice were intraperitoneally injected with cisplatin (20 mg/kg) to induce AKI; - Chronic kidney injury (CKI) model: 8-week-old male C57BL/6 mice were intraperitoneally injected with streptozotocin (STZ, 50 mg/kg) once daily for 5 consecutive days to induce type 1 diabetes, which progresses to diabetic nephropathy (CKI) within 12 weeks [3]
2. Drug preparation and administration: - CAY10603 was dissolved in DMSO (10% v/v) and diluted with physiological saline to a final concentration of 1 mg/mL (DMSO final concentration <5%). The vehicle control was 5% DMSO in physiological saline [3]
- AKI model mice: CAY10603 was administered intraperitoneally at 10 mg/kg once daily for 5 days, starting 1 hour after cisplatin injection; - CKI model mice: CAY10603 was administered intraperitoneally at 10 mg/kg 5 times per week for 12 weeks, starting 1 week after the last STZ injection [3]
3. Sample collection and analysis: - Mice were euthanized at the end of treatment. Blood was collected via orbital plexus to measure serum creatinine (Scr) and blood urea nitrogen (BUN) using commercial biochemical kits; - Kidneys were removed: one kidney was fixed in 4% paraformaldehyde for histopathological staining (HE staining for AKI, Masson trichrome staining for CKI), and the other kidney was dissected into renal cortex for Western blot analysis [3]

1. In vitro cytotoxicity to normal cells: CAY10603 (0.1–10 μM, 48 hours) had no significant effect on the viability of normal HK-2 cells (without injury induction), with a cell viability of >90% (MTT assay) [3]
2. In vivo safety: - During the 5-day treatment for AKI and 12-week treatment for CKI, CAY10603-treated mice showed no significant changes in body weight (e.g., CKI model: final body weight 24.5 ± 1.3 g vs. 23.8 ± 1.5 g in vehicle control group); - Serum levels of liver function markers (ALT, AST) were within the normal range and not significantly different from the vehicle control group (ALT: 26 ± 4 U/L vs. 24 ± 3 U/L; AST: 38 ± 5 U/L vs. 36 ± 4 U/L) [3]

[1]. Use of the nitrile oxide cycloaddition (NOC) reaction for molecular probe generation: a new class of enzyme selective histone deacetylase inhibitors (HDACIs) showing picomolar activity at HDAC6. J Med Chem. 2008 Aug 14;51(15):4370-3.

[2]. HDAC6 promotes cell proliferation and confers resistance to gefitinib in lung adenocarcinoma. Oncol Rep. 2016 Jul;36(1):589-97.

[3]. Inhibition of HDAC6 with CAY10603 alleviates acute and chronic kidney injury by suppressing the ATF6 branch of UPR. Arch Biochem Biophys . 2024 Jun:756:110009.

N-[4-[3-[[[7-(hydroxyamino)-7-oxoheptyl]amino]-oxomethyl]-5-isoxazolyl]phenyl]carbamic acid tert-butyl ester is a carbamate ester.

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A series of hydroxamate based HDAC inhibitors containing a phenylisoxazole as the CAP group has been synthesized using nitrile oxide cycloaddition chemistry. An HDAC6 selective inhibitor having a potency of approximately 2 picomolar was identified. Some of the compounds were examined for their ability to block pancreatic cancer cell growth and found to be about 10-fold more potent than SAHA. This research provides valuable, new molecular probes for use in exploring HDAC biology.[1]

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Histone deacetylases (HDACs) are promising targets for cancer therapy, and first-generation HDAC inhibitors are currently in clinical trials for the treatment of cancer patients. HDAC6, which is a key regulator of many signaling pathways that are linked to cancer, has recently emerged as an attractive target for the treatment of cancer. In the present study, HDAC6 was found to be overexpressed in lung adenocarcinoma cell lines and was negatively correlated with the prognosis of patients with lung adenocarcinoma. Overexpression of HDAC6 promoted the proliferation of lung adenocarcinoma cells in a deacetylase activity-dependent manner. HDAC6 overexpression conferred resistance to gefitinib via the stabilization of epidermal growth factor receptor (EGFR). The inhibition of HDAC6 by CAY10603, a potent and selective inhibitor of HDAC6, inhibited the proliferation of lung adenocarcinoma cells and induced apoptosis. CAY10603 downregulated the levels of EGFR protein, which in turn inhibited activation of the EGFR signaling pathway. Moreover, CAY10603 synergized with gefitinib to induce apoptosis of the lung adenocarcinoma cell lines via the destabilization of EGFR. Taken together, our results suggest that the inhibition of HDAC6 may be a promising strategy for the treatment of lung adenocarcinoma.[2]

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A series of 2-phenylthiazole analogues were designed and synthesized as potential histone deacetylase 6 (HDAC6) inhibitors based on compound 12c (an HDAC6/tubulin dual inhibitor discovered by us recently) and CAY10603 (a known HDAC6 inhibitor). Among them, compound XP5 was the most potent HDAC6 inhibitor with an IC50 of 31 nM and excellent HDAC6 selectivity (SI = 338 for HDAC6 over HDAC3). XP5 also displayed high antiproliferative activity against various cancer cell lines including the HDACi-resistant YCC3/7 gastric cancer cells (IC50 = 0.16-2.31 μM), better than CAY10603. Further, XP5 (50 mg/kg) exhibited significant antitumor efficacy in a melanoma tumor model with a tumor growth inhibition (TGI) of 63% without apparent toxicity. Moreover, XP5 efficiently enhanced the in vivo antitumor immune response when combined with a small-molecule PD-L1 inhibitor, as demonstrated by the increased tumor-infiltrating lymphocytes and reduced PD-L1 expression levels. Taken together, the above results suggest that XP5 is a promising HDAC6 inhibitor deserving further investigation.J Med Chem. 2022 Feb 10;65(3):2434-2457.

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1. Mechanism of action: CAY10603 alleviates acute and chronic kidney injury by selectively inhibiting HDAC6. HDAC6 inhibition increases Ac-α-tubulin levels, which reduces the nuclear translocation of ATF6, thereby suppressing the activation of the ATF6 branch of the unfolded protein response (UPR). This leads to decreased endoplasmic reticulum stress, reduced cell apoptosis, and inhibited inflammation and fibrosis in renal tissues [3]
2. Research background: HDAC6 is upregulated in renal injury models (e.g., cisplatin-induced AKI, diabetic CKI), and activation of the ATF6-UPR pathway promotes renal cell death and fibrosis. CAY10603 as a selective HDAC6 inhibitor provides a potential therapeutic strategy for kidney injury [3]
3. Clinical status: The 2024 publication indicates that CAY10603 is in preclinical research for kidney injury; no clinical trials, FDA approval, or indication-related information was reported [3]

C22H30N4O6

446.5

446.216

C, 59.18; H, 6.77; N, 12.55; O, 21.50

1045792-66-2

24951314

White to light yellow solid powder

1.2±0.1 g/cm3

1.563

1.94

142.79

4

7

12

32

616

0

O(C(N([H])C1C([H])=C([H])C(=C([H])C=1[H])C1=C([H])C(C(N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(N([H])O[H])=O)=O)=NO1)=O)C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H]

WWGBHDIHIVGYLZ-UHFFFAOYSA-N

InChI=1S/C22H30N4O6/c1-22(2,3)31-21(29)24-16-11-9-15(10-12-16)18-14-17(26-32-18)20(28)23-13-7-5-4-6-8-19(27)25-30/h9-12,14,30H,4-8,13H2,1-3H3,(H,23,28)(H,24,29)(H,25,27)

tert-butyl N-[4-[3-[[7-(hydroxyamino)-7-oxoheptyl]carbamoyl]-1,2-oxazol-5-yl]phenyl]carbamate

CAY-10603; CAY10603; BML-281; tert-Butyl (4-(3-((7-(hydroxyamino)-7-oxoheptyl)carbamoyl)isoxazol-5-yl)phenyl)carbamate; CAY-10603; tert-butyl N-[4-[3-[[7-(hydroxyamino)-7-oxoheptyl]carbamoyl]-1,2-oxazol-5-yl]phenyl]carbamate; CHEMBL511749; compound 3 [PMID: 18642892]; CAY 10603

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.60 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.60 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.60 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 5% DMSO+50% PEG 300+ddH2O: 9mg/mL

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配方 5 中的溶解度: 5 mg/mL (11.20 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液； 需要超声波加热并加热至 42°C。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。