DHODH (dihydroorotate dehydrogenase; IC50 = 5.2 nM)

Brequinar是一种具有抗肿瘤特性的二氢乳清酸脱氢酶/DHODH抑制剂。氯喹抑制二氢乳清酸脱氢酶，从而阻断嘧啶的从头生物合成。
Brequinar 的 EC50 为 17 nM，可将病毒后代的形成减少 90% 以上。此外，Brequinar (5 μM) 还可抑制其他正痘病毒并阻断病毒 DNA 复制。 Brequinar 对病毒周期的后期有很强的影响，尽管它对早期病毒基因的表达没有影响[1]。 Brequinar 在 CFI 测试中的 EC50 为 78 nM，以剂量依赖性方式降低包膜蛋白合成量和病毒滴度。 Brequinar (5 μM) 可防止病毒 RNA 的合成。 Brequinar 具有抗病毒作用，但嘧啶可以中和它。在细胞培养中，可以选择对布喹那具有抗性的病毒。 WT 和 NS5 突变体复制子的荧光素酶活性均被 Bequinar (5 μM) 抑制[2]。布喹那钠成功抑制了 PyNTP 的升高。 Brequinarodium 的 IC50 为 0.26 μM，可显着抑制细胞增殖。 Brequinarodium 抑制 p56lck 自磷酸化，IC50 为 70 μM； 25、50和100μM布喹那钠的相应抑制值为39%、41%和60%。此外，在 IC50 为 70 μM 时，贝奎那钠可防止 p56lck 对外源底物组蛋白 2B 的磷酸化；在 25、50、100 和 200 μM 时，抑制分别为 10%、43%、59% 和 86%。 Brequinar Brequinar钠的IC50为105 μM，在25、50、100和200 μM时分别抑制p59fyn的自磷酸化0、17、48和65%。此外，在 IC50 为 20 μM 时，Brequinarodium 抑制 p59fyn 对组蛋白 2B 的磷酸化；在 10、25、50、100 和 200 μM 时，相应的抑制率为 26、54、79、83 和 84%。

如前所述 (20) 将 BALB/c 心脏移植到 C3H 小鼠中。 BQR通过腹腔注射每天一次，而尿苷每天两次。使用微量血细胞比容毛细管通过眼眶静脉对小鼠放血，并将血液以550 3 g离心10分钟。用微量血细胞比容毛细管读数器测定堆积细胞体积的百分比。所有小鼠在接受最后一剂 BQR 或尿苷后 4 小时处死。

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Brequinar/BQR诱导BALB/c小鼠贫血的能力可通过联合给药尿苷来阻止。相反，BQR的免疫抑制活性不受相同剂量尿苷的影响。用BQR治疗的小鼠，骨髓中的PyN水平下降，而脾脏中没有。这些观察结果表明，BQR诱导贫血是由于位于骨髓的造血干细胞中细胞内PyN的耗尽。他们还表明，BQR的免疫抑制机制可能仅少量依赖于位于周围的淋巴细胞中细胞内PyN的消耗。我们报道了BQR的一种新活性:抑制酪氨酸磷酸化，并假设免疫抑制活性可能部分归因于BQR在淋巴细胞中抑制酪氨酸磷酸化的这种意想不到的能力。[2]

BQR是细胞二氢乳清酸脱氢酶的有效抑制剂，二氢乳清酸酯脱氢酶是一种从头嘧啶生物合成途径的酶。在存在0.5μM BQR的情况下感染使病毒子代产量减少了>90%，显示EC（50）（抑制50%病毒复制所需的药物浓度）为0.017μM。BQR也以类似的水平抑制其他正痘病毒的复制。在药物存在的情况下，病毒早期蛋白质积累到控制水平，而晚期基因表达严重受损。这一结果通过间接免疫荧光测定和在病毒启动子控制下报告基因的时间调节表达的分析得到证实。两种测定都显示晚期基因表达受到近90%的抑制。BQR还阻断了病毒DNA的复制，这是随后抑制病毒晚期基因表达的原因。当用尿苷（URD）和BQR共同处理感染细胞时，病毒DNA复制、晚期基因表达和感染后代产生的消融恢复到对照水平。这些数据表明，BQR通过消耗细胞嘧啶库来靶向病毒DNA合成，当将URD添加到细胞培养物中时，挽救途径绕过了嘧啶库[1]。

细胞内嘧啶核苷酸（PyN）可以由谷氨酰胺、CO2和ATP从头合成，也可以从预先形成的嘧啶核苷中回收。布雷奎纳钠（BQR）的抗增殖和免疫抑制活性被认为是由于抑制了二氢乳清酸脱氢酶的活性，从而抑制了从头嘧啶的合成。在这里，我们描述嘧啶核苷尿苷对BQR的抗增殖和免疫抑制活性的影响。尿苷逆转了Con A刺激的T细胞中PyN水平的体外降低和低浓度BQR（＜或＝65μM）对细胞增殖的抑制。然而，尿苷不能逆转高浓度BQR（>=65μM）的影响[2]。

Eight to ten-week-old female BALB/C mice were randomized and not blinded. Molnupiravir was resuspended in corn oil and 10% DMSO, with dosing twice a day as oral gavage. Brequinar was resuspended in 10% DMSO and sterile saline, with dosing daily as intraperitoneal injection. Our pharmacokinetic (PK) studies showed an approximately 10 h half-life of Brequinar, and dosing was either started at 12 h before infection or 24 h after infection as indicated.[3]

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Method 1: Brequinar sodium was dissolved in 0.9% NaCl for i.p. injections and in distilled water for in vitro studies. [1] Method 2: Brequinar sodium was formulated as a 10 mg/mL solution in saline. [2]

Mice

Pharmacokinetic (PK) studies showed an approximately 10 h half-life of Brequinar. [3]

[1]. Int J Antimicrob Agents. 2011 Nov;38(5):435-41.
[2]. J Immunol. 1998 Jan 15;160(2):846-53.

[3]. Nature. 2022 Apr;604(7904):134-140.

Brequinar sodium is an organic sodium salt of brequinar. It has a role as an anticoronaviral agent, an antineoplastic agent, an antiviral agent, an EC 1.3.5.2 [dihydroorotate dehydrogenase (quinone)] inhibitor, an immunosuppressive agent, a pyrimidine synthesis inhibitor and an antimetabolite. It contains a brequinar(1-).
Brequinar Sodium is the sodium salt form of Brequinar. Brequinar inhibits the enzyme dihydroorotate dehydrogenase, thereby blocking de novo pyrimidine biosynthesis. This agent may also enhance the in vivo antitumor effect of antineoplastic agents such as 5-FU. (NCI04)

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In the present work, the antiviral activity of brequinar (BQR) against the replication of Cantagalo virus was evaluated. BQR is a potent inhibitor of cellular dihydroorotate dehydrogenase, an enzyme of the de novo pyrimidine biosynthetic pathway. Infection in the presence of 0.5μM BQR reduced virus progeny production by >90%, revealing an EC(50) (drug concentration required to inhibit 50% of virus replication) of 0.017μM. Replication of other orthopoxviruses was also inhibited by BQR at similar levels. In the presence of the drug, virus early proteins accumulated to control levels, whereas late gene expression was severely impaired. This result was confirmed by indirect immunofluorescence assays and analysis of time-regulated expression of a reporter gene under the control of a virus promoter. Both assays revealed nearly 90% inhibition of late gene expression. BQR also blocked virus DNA replication, which accounted for the subsequent inhibition of virus late gene expression. The ablation of virus DNA replication, late gene expression and infectious progeny production was restored to control levels when infected cells were co-treated with uridine (URD) and BQR. These data demonstrated that BQR targeted virus DNA synthesis by depleting the cellular pyrimidine pool, which was bypassed by the salvage pathway when URD was added to the cell cultures. [1]

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Intracellular pyrimidine nucleotides (PyN) can be synthesized de novo from glutamine, CO2, and ATP, or they can be salvaged from preformed pyrimidine nucleosides. The antiproliferative and immunosuppressive activities of brequinar sodium (BQR) are thought to be due to the inhibition of the activity of dihydroorotate dehydrogenase, which results in a suppression of de novo pyrimidine synthesis. Here we describe the effects of the pyrimidine nucleoSide, uridine, on the antiproliferative and immunosuppressive activities of BQR. In vitro reduction of PyN levels in Con A-stimulated T cells and inhibition of cell proliferation by low concentrations of BQR (< or =65 microM) are reversed by uridine. However, uridine is unable to reverse the effects of high concentrations of BQR (> or =65 microM). The ability of BQR to induce anemia in BALB/c mice is prevented by the coadministration of uridine. In contrast, the immunosuppressive activity of BQR is unaffected by similar doses of uridine. PyN levels in the bone marrow, but not in the spleen, are depressed in mice treated with BQR. These observations suggest that the induction of anemia by BQR is due to depletion of intracellular PyN in hemopoietic stem cells located in the bone marrow. They also suggest that the mechanism of immunosuppression by BQR may be only marginally dependent on depletion of intracellular PyN in lymphocytes located in the periphery. We report a novel activity of BQR: inhibition of tyrosine phosphorylation, and hypothesize that the immunosuppressive activity may be due, in part, to this unsuspected ability of BQR to inhibit tyrosine phosphorylation in lymphocytes. [2]

C23H14F2NNAO2

397.36

397.089

C, 69.52; H, 3.55; F, 9.56; N, 3.53; Na, 5.79; O, 8.05

96201-88-6

Brequinar;96187-53-0

23663964

Typically exists as White to off-white solid at room temperature

550.9ºC at 760mmHg

>360°

287ºC

5.853

50.19

0

5

3

29

557

0

[Na].O=C(C1C(C)=C(C2C=CC(C3C(F)=CC=CC=3)=CC=2)N=C2C=1C=C(C=C2)F)O

PZOHOALJQOFNTB-UHFFFAOYSA-M

InChI=1S/C23H15F2NO2.Na/c1-13-21(23(27)28)18-12-16(24)10-11-20(18)26-22(13)15-8-6-14(7-9-15)17-4-2-3-5-19(17)25/h2-12H,1H3,(H,27,28)/q+1/p-1

Sodium 6-Fluoro-2-(2'-fluoro[1,1'-biphenyl]-4-yl)-3-methyl-4-quinolinecarboxylate

Dup785 NSC-368390 Dup 785 NSC368390 Dup-785 Brequinar SodiumBipenquinate

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。