Impurity spectrum analysis of Tenofovir Disoproxil: selection of seven major categories of impurities and reference substances

A nucleotide drug that changes the antiviral treatment landscape

Tenofovir Disoproxil (CAS 201341-05-1)It is a nucleotide reverse transcriptase inhibitor (NtRTI) developed by Gilead of the United States.It is one of the few antiviral drugs approved for both HIV (human immunodeficiency virus) infection and chronic hepatitis B (HBV), and has long been the first-line drug recommended by many treatment guidelines.

Tenofovir Disoproxil

Its commonly used clinical salt form isTenofovir Disoproxil Fumarate (TDF, CAS 202138-50-9), trade name "Viread". Various compound preparations composed of TDF, emtricitabine, efavirenz and other drugs are also an important part of the anti-HIV treatment plan.

Brief description of mechanism of action:After oral absorption, tenofovir disoproxil is rapidly hydrolyzed to the active parent tenofovir (Tenofovir, PMPA, CAS 147127-20-6), which is phosphorylated by kinases in cells to generate the active metabolite tenofovir diphosphate, which competes with the natural substrate deoxyadenosine 5'-triphosphate and is incorporated into the viral DNA chain. Due to the lack of 3′-OH groups in its molecule, DNA chain elongation is hindered, thereby blocking viral replication.

"Prodrug design" is the key to efficacy and also the difficulty in impurity control

tenofovirIt is a highly polar, charged acyclic nucleotide phosphonic acid analogue with very low oral bioavailability. In order to solve the absorption problem, the developers esterified its phosphonic acid group into a neutral and more lipophilic bis(isopropoxycarbonyloxymethyl) ester structure.This is the origin of "tenofovir disoproxil disoproxil (Bis(POC)-PMPA)". After the drug enters the body, it is hydrolyzed step by step to release active tenofovir.

This clever prodrug design brings good oral efficacy, but it also poses two challenges to quality control:

• Ester bonds are easily hydrolyzed:The carbonate structure is prone to hydrolysis under conditions of moisture, heat or pH changes, producing a series of degradation impurities (such as monoesters, parent tenofovir, etc.).

• The synthesis route is long and the reagents are active:Multi-step reactions such as the construction of adenine fragments, phosphonate esterification, and introduction of chiral centers will introduce starting materials, intermediates, by-products, and potentially genotoxic impurities.

Because of this, the impurity profile of tenofovir disoproxil is relatively complex. In accordance with the requirements of ICH Q3A/Q3B, M7, Chinese Pharmacopoeia, USP, EP and other requirements, the qualitative positioning, content determination and methodological verification of relevant substances cannot be carried out without impurity reference standards (Reference Standards) with clear structures and reliable sources as "rulers". Impurity research has therefore become an unavoidable core task in the development, registration application and quality release of raw materials and preparations.

Tenofovir disoproxil impurity "family tree"

Combining the prodrug characteristics and synthesis route of tenofovir disoproxil, its related impurities can be roughly classified into the following seven categories:Corresponding standards for each category can be found in the CATO catalog:

(The above product numbers and CAS numbers are quoted from the CATO tenofovir disoproxil product catalog for positioning reference.)

For all the above impurity categories, CATO can provide more than 140 kinds of tenofovir/tenofovir disoproxil related reference substances, covering the full spectrum from process impurities, degradation impurities, isomers, oligomers to isotope internal standards, with specifications ranging from milligram level to gram level. CATO has ISO 17034 standard material producer qualifications (dual accreditation of CNAS RM0030 and ANAB AR-2832), providing support for value traceability and inter-batch consistency.