Azolo[1,5-a]pyrimidines and Their Condensed Analogs with Anticoagulant Activity

Hypercytokinemia, or cytokine storm, is one of the severe complications of viral and bacterial infections, involving the release of abnormal amounts of cytokines, resulting in a massive inflammatory response. Cytokine storm is associated with COVID-19 and sepsis high mortality rate by developing epithelial dysfunction and coagulopathy, leading to thromboembolism and multiple organ dysfunction syndrome. Anticoagulant therapy is an important tactic to prevent thrombosis in sepsis and COVID-19, but recent data show the incompatibility of modern direct oral anticoagulants and antiviral agents. It seems relevant to develop dual-action drugs with antiviral and anticoagulant properties. At the same time, it was shown that azolo[1,5-a]pyrimidines are heterocycles with a broad spectrum of antiviral activity. We have synthesized a new family of azolo[1,5-a]pyrimidines and their condensed polycyclic analogs by cyclocondensation reactions and direct CH-functionalization and studied their anticoagulant properties. Five compounds among 1,2,4-triazolo[1,5-a]pyrimidin-7-ones and 5-alkyl-1,3,4-thiadiazolo[3,2-a]purin-8-ones demonstrated higher anticoagulant activity than the reference drug, dabigatran etexilate. Antithrombin activity of most active compounds was confirmed using lipopolysaccharide (LPS)-treated blood to mimic the conditions of cytokine release syndrome. The studied compounds affected only the thrombin time value, reliably increasing it 6.5–15.2 times as compared to LPS-treated blood.

Azolo[1,5-a]pyrimidines and Their Condensed Analogs with Anticoagulant Activity

Hypercytokinemia, or cytokine storm, is one of the severe complications of viral and bacterial infections, involving the release of abnormal amounts of cytokines, resulting in a massive inflammatory response. Cytokine storm is associated with COVID-19 and sepsis high mortality rate by developing epithelial dysfunction and coagulopathy, leading to thromboembolism and multiple organ dysfunction syndrome. The anticoagulant therapy is an important tactic to prevent thrombosis in sepsis and COVID-19, but recent data show the incompatibility of modern direct oral anticoagulants and antiviral agents. It seems relevant to develop dual-action drugs with antiviral and anticoagulant properties. At the same time it was shown that azolo[1,5-a]pyrimidines are heterocycles with a broad spectrum of antiviral activity. We have synthesized a new family of azolo[1,5-a]pyrimidines and their condensed polycyclic analogs by cyclocondensation reactions and direct CH-functionalization and studied their anticoagulant properties. Five compounds among 1,2,4-triazolo[1,5-a]pyrimidin-7-ones and 5-alkyl-1,3,4-thiadiazolo[3,2-a]purin-8-ones demonstrated higher anticoagulant activity than the reference drug, dabigatran etexilate. Antithrombin activity of lead compounds was confirmed using lipopolysaccharide (LPS) treated blood to mimic conditions of cytokine release syndrome. The studied compounds affected only the thrombin time value, reliably increasing it 6.5–15.2 times as compared to LPS-treated blood.

Retrospective analysis of the effectiveness of a reduced dose of idarucizumab in dabigatran reversal

Background The recommended dose of idarucizumab, the specific reversal agent for dabigatran etexilate, is 5g. However, published data showed biochemical reversal after an initial 2.5g dose. Objectives This study aims to retrospectively compare the clinical effectiveness of 2.5g and 5g doses of idarucizumab used in dabigatran reversal in three hospitals in Auckland, New Zealand. Methods All patients receiving idarucizumab for dabigatran reversal between 1st April 2016 and 31st December 2018 were included. The primary outcome was the likelihood of receiving a second dose of idarucizumab during the same admission. Secondary outcomes included normalisation of coagulation profiles; and 30-day thrombotic, bleeding and mortality rates. Results Of 329 patients included, 206 received an upfront 2.5g dose and 123 received a 5g dose. The median age was 78 years and median creatinine clearance was 50mL/min. Most patients (62.6%) required idarucizumab for an urgent procedure, while 37.4% presented with bleeding. A 2.5g dose was not associated with an increased rate of receiving a second dose (OR 0.686, 95% CI 0.225-2.090). A similar proportion of patients in each group achieved a normal APTT (73.8% vs 80.0%, p=0.464) and dTCT (95.9% vs 91.4%, p=0.379) following idarucizumab infusion. There was no increase in the rate of death (OR 0.602, 95% CI 0.292-1.239), thrombosis (OR 0.386, 95% CI 0.107-1.396) or bleeding (OR 0.96, 95% CI 0.27-3.33) in the 2.5g dose group compared to the 5g dose group. Conclusions An initial 2.5g dose of idarucizumab appears effective for dabigatran reversal in the real-world setting.

Development and validation of an analytical method for the determination of direct oral anticoagulants (DOAC) and the direct thrombin-inhibitor argatroban by HPLC–MS/MS

Since direct oral anticoagulants (DOAC) are administered frequently to an elderly, co-morbid population, medical emergencies including trauma, acute bleeding or organ failure are not uncommon. In these situations, the type, dosage or the time of last intake of anticoagulants is often unknown and single substance analysis by functional tests is only possible if the substance contained in the sample is known. A reliable and validated toxicology screen of DOAC and argatroban would be helpful inform not only attending physicians in the emergency department but also law enforcement and courts of justice. After precipitation with acetone, HPLC separation was achieved on a Phenomenex Luna Pentafluorophenyl Colum using acetonitrile–water (90:10, v/v) as mobile phase system. Detection was performed using a 3200 Q Trap mass spectrometer (AB Sciex). For analysis MRM Scans (MS/MS) with positive ionization were chosen. The method was validated for blank serum as the matrix of choice. Limits of detection are between 0.5 and 1.0 ng/mL, limits of quantification are between 1.9 and 3.6 ng/mL and recoveries are above 60%. The applicability of the method was demonstrated by the determination of DOAC in body fluids from forensic cases and in therapeutic drug monitoring. The rapid simultaneous detection and quantification of apixaban, argatroban, dabigatran etexilate, dabigatran, edoxaban and rivaroxaban in body fluids by HPLC–MS/MS closes an important gap in emergency toxicology.