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In vitro anticoagulant activity [1]
All target compounds were evaluated for their anticoagulant activity by using human and rabbit plasma in vitro. The results were expressed as prothrombin time (PT) and activated partial thromboplastin time (APTT). PT measures the effect of a compound on the extrinsic pathway of coagulation, whereas APTT represents the effect on the intrinsic pathway. The results were expressed as EC2x values and are summarized in Table 1. The EC2x values in Table 1 are the average of at least three independent experiments.

As shown in Table 1, most of the target compounds exhibited moderate to excellent activity, with the EC2x value from 30 to 0.5 μM, wherein the activity of compounds 14a (PT = 0.5, APTT = 0.8), 16 (PT = 0.8, APTT = 1.9), 18c (PT = 1.0, APTT = 2.3), 26c (PT = 1.1, APTT = 2.4), 35a (PT = 0.9, APTT = 0.9), and 35b (PT = 0.9, APTT = 1.1) was comparable to that of apixaban (PT = 0.8, APTT = 0.9). Particularly, compound 14a was the most potent candidate for further research in these series. Furthermore, the anticoagulant activity in rabbit and human plasma was correlative.

During the investigation of the C-3 pyrazole position, as illustrated in Table 1, 14a (PT = 0.5, APTT = 0.8) with unsubstituted 1,2,4-triazole showed superior anticoagulant activity. Unfortunately, different electron-withdrawing groups (EWGs) in the methoxyl position decreased the activity obviously. The order of anticoagulant activity was –OCH3 (14a) > –F (14d) > –OCHF2 (14c) > –Cl (14e) > –OCF3 (14b) > –Br (14f), suggesting the importance of methoxyl as an electron-donating group (EDG) in the P1 ligand.

Upon comparing the isomers 15 and 16, significant differences were observed between 2-N-methyl and 1-N-methyl in anticoagulant activity. By switching from the N-methyl to C-methyl position, 18a was produced by introducing 5-C-methyl into 1,2,4-triazole. The activity of 18a was between that of compounds 15 and 16 (16 > 18a > 15). Subsequently, a series of derivatives 18b–18e were synthesized with different substituents at 5-C. They showed a decrease in anticoagulant potency except for compound 18c with t-butyl (PT = 1.0, APTT = 2.3), indicating that the 5-C-substituted EDG and steric effects were factors that could not be neglected.

Compounds 21a–21d were also synthetized to increase the hydrophilicity or solubility by adding an alkaline hydrophilic group. All of them displayed decreased potency. For increasing hydrogen bond action, 5-oxo-substituted 1,2,4-triazole with 4-N-phenyl was introduced into the C-3 pyrazole to obtain compounds 23a, 23b, and 23c. The pharmacological data indicated that the activity was lost completely (PT > 12, APTT > 20). A plausible explanation for this is that the steric hindrance produced by the introduction of large groups influenced the activity.

Further studies were performed to examine the effect of different nitrogenous heterocyclics on the C-3 pyrazole. Compounds 26a, 26b, and 26c were synthetized with alkaline-substituted pyrrole moieties. The biological data showed that compounds 26c (PT = 1.1, APTT = 2.4), 26a (PT = 1.5, APTT = 3.5), and 26b (PT = 1.6, APTT = 3.7) possessed high anticoagulant activity.

As illustrated in Table 1, the introduction of dihydroimidazol and tetrahydropyrimidine groups into the hydrophobic pocket (P4 region) could improve the anticoagulant activity, such as compounds 35a (PT = 0.9, APTT = 0.9) and 35b (PT = 0.9, APTT = 1.1). More studies on the P4 moiety are under way.

The SARs based on the EC2x values in Table 1 showed that the hydrogen bond action and size of the nitrogenous heterocyclic in P2 and the lipophilic region in P4 were responsible for the anticoagulant activity.

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In vitro FXa enzymatic assays and in vivo antithrombotic effecty [1]
Based on the anticoagulant potency, as shown in Table 1, the six tested compounds 14a, 16, 18c, 26c, 35a, and 35b showed excellent anticoagulant activity. Therefore, these compounds, at their IC50 values, were tested against human FXa in vitro, as listed in Table 2. The fitting curve, indicating the percent of inhibition, and the curve detailing the IC50 values of 14a and 35b are summarized in Figure 4, Figure 5, respectively. Wherein compound 14a displays the most potent activity against human FXa with an IC50 value of 0.15 μM and 99% inhibition rate in the rat venous thrombosis test, which is superior to that of apixaban.

In vivo antithrombotic effect [1]
Based on the anticoagulant potency, as shown in Table 1, the six tested compounds 14a, 16, 18c, 26c, 35a, and 35b showed excellent anticoagulant activity. Therefore, these compounds, at their IC50 values, were tested against rat venous thrombosis in vivo, as listed in Table 2. The fitting curve, indicating the percent of inhibition, and the curve detailing the IC50 values of 14a and 35b are summarized in Figure 4, Figure 5, respectively. Wherein compound 14a displays the most potent activity against human FXa with an IC50 value of 0.15 μM and 99% inhibition rate in the rat venous thrombosis test, which is superior to that of apixaban.

In vitro coagulation assays [1]
PT and APTT were measured using commercially available kits. Blood was obtained from healthy human volunteers or rabbits and anticoagulated with 3.8% sodium citrate. Plasma was obtained after centrifugation at 2000 g for 10 min. An initial stock solution of the inhibitor was prepared in DMSO. Subsequent dilutions were done. Clotting time was determined using the control plasma and plasma containing five to seven different concentrations of the inhibitor. PT measurement was performed in a temperature-controlled automated coagulation device using a Thromborel-S kit according to the reagent instructions. Determinations at each plasma concentration were done in duplicate.

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In vitro human FXa inhibition assays [1]
The potent inhibitory activity of six test compounds against human FXa was evaluated by using chromogenic substrate in 96-well microtiter plate. The enzymatic action of human factor Xa was measured using the conversion of a chromogenic substrate specific for FXa. FXa cleaved p-nitroaniline from the chromogenic substrate (Table 4).

In vivo experimental vein thrombosis [1]
Vein thrombosis was induced according to Reyers et al.25 with modifications. Briefly, the animals were anesthetized with pentobarbital (40 mg/kg, ip) and the abdomen was opened. The venacava inferior was carefully separated from surrounding tissues and ligated tightly with a cotton thread just below the left renal vein. The abdomen was then closed with a double layer of sutures. After 2 h, the abdomen was reopened, and the vena cava was dissected longitudinally and the formed thrombus was removed. The obtained thrombi were kept at 37 °C for 24 h and after this time, their dry weight was measured.

[1]. Design, synthesis, and structure-activity relationship of novel and effective apixaban derivatives as FXa inhibitors containing 1,2,4-triazole/pyrrole derivatives as P2 binding element. Bioorg Med Chem. 2016 Nov 1;24(21):5646-5661.

Four series of novel and potent FXa inhibitors possessing the 1,2,4-triazole moiety and pyrrole moiety as P2 binding element and dihydroimidazole/tetrahydropyrimidine groups as P4 binding element were designed, synthesized, and evaluated for their anticoagulant activity in human and rabbit plasma in vitro. Most compounds showed moderate to excellent activity. Compounds 14a, 16, 18c, 26c, 35a, and 35b were further examined for their inhibition activity against human FXa in vitro and rat venous thrombosis in vivo. The most promising compound 14a, with an IC50 (FXa) value of 0.15μM and 99% inhibition rate, was identified for further evaluation as an FXa inhibitor.[1]

In this study, four series of novel potent FXa inhibitors based on apixaban were designed and synthesized. The pharmacological data indicated that several compounds (14a, 16, 18c, 26c, 35a, and 35b) exhibited moderate to excellent anticoagulant potency in human and rabbit plasma in vitro. The SARs, based on the EC2x values shown in Table 1, showed that the hydrogen bond action and size of the nitrogenous heterocyclic in P2 and the lipophilic region in P4 were responsible for the anticoagulant activity. Eventually, compound 14a possessing a novel scaffold was selected for further exploration owing to its pronounced enzymatic anticoagulant activity with an IC50 value of 0.15 μM in vitro and 99% inhibition rate in rat venous thrombosis test in vivo. The docking model indicated that compound 14a formed a hydrogen bond with Glu146, and 1,2,4-triazole provided a much more suitable size (5-membered triazole ring) accommodating the position of P2 compared with apixaban. [1]

C27H28N4O5

488.54

488.205

503614-91-3

22240488

Solid powder

1.3±0.1 g/cm3

758.7±60.0 °C at 760 mmHg

412.6±32.9 °C

0.0±2.6 mmHg at 25°C

1.658

2.47

0

6

7

36

808

0

CCOC(C1=NN(C2C=CC(OC)=CC=2)C2C(N(C3C=CC(N4CCCCC4=O)=CC=3)CCC1=2)=O)=O

PULNLYVCJSOXKS-UHFFFAOYSA-N

InChI=1S/C27H28N4O5/c1-3-36-27(34)24-22-15-17-30(19-9-7-18(8-10-19)29-16-5-4-6-23(29)32)26(33)25(22)31(28-24)20-11-13-21(35-2)14-12-20/h7-14H,3-6,15-17H2,1-2H3

ethyl 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-4,5-dihydropyrazolo[3,4-c]pyridine-3-carboxylate

503614-91-3; ethyl 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxylate; 1-(4-Methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxylic acid ethyl ester; Apixaban Impurity 9; APIXABAN V; ethyl 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-4,5-dihydropyrazolo[3,4-c]pyridine-3-carboxylate; Apixaban Ethyl Ester; MFCD18072444;

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)