Controlling a cohort Understanding the risk of nitrosamines within drug substance synthesis Dr Michael Burns Senior Scientist michael.burns@lhasalimited.org
Outline • Purge approach for nitrosamine risk assessments • Exploring potential reactivity of nitrosamines • Formation of N -nitroso compounds (NOCs)
Purge approach for nitrosamine risk assessments
Purge assessments and risk assessments • Industry were faced with a near insurmountable task to review all products within 6 months (March 2020). • Deadline was extended by 6 months to October 2020. • Batch testing for the presence of nitrosamines within all drug substances is problematic • Insufficient worldwide capacity for testing of appropriate sensitivity • In the short term identifying highest potential risk products is key. • Industry group EFPIA have been working to establish a workflow to address this issue. • EFPIA and EMA are engaging to get to a point of agreement. • If there are no nitrosating agents or secondary/tertiary amines present within the synthetic route (as reagents or by-products) then risk is deemed low/non-existent.
What if there is a potential risk? • If amines and nitrosating agents are present, then a risk does exist, but not all risks are high. • Both components must be present at high enough levels within the same step to create a significant risk of formation. • Where present together, conditions must be conducive to nitrosamine formation (e.g. acidic conditions) • Purge assessments can be used in two ways to determine the risk in line with ICH M7 control options. • Is there a genuine risk of nitrosamine formation? • Will potential nitrosamines persist into the API?
De-risking Candesartan (AZ) • Candesartan is a drug marketed by AZ • Risk is present from potential amines resulting from triethylamine and DMF degradation. • Purge assessment of these impurities indicates there is no realistic possibility of NaNO 2 being present in the same stage as an amine. • Even if formed, a reasonable potential to purge also exists in the subsequent stages.
Understanding purge In-depth understanding of the process conditions is vital, as this allows appropriate use of purge values.
Purge Ratio • The use of the purge ratio (PR) has been widely adopted to define the regulatory reporting expectations for purge calculations, with further conservatism built in. • E.g. Where PR > 1000 - very little support is required to back up an option 4 approach • How can the purge ratio be utilised within the nitrosamine risk assessment? 8 Barber et al, Regul. Toxicol. Pharmacol., 2017, 90, 22-28
Utilising Purge Ratio • Nitrosamine risk is only present if they can be formed in sufficient quantities to exceed permissible levels. • Principles of the purge ratio can be applied to the components required to generate a nitrosamine to act as a guide to the risk of formation at a concerning level Predicted Purge Predicted Purge Purge Ratio Purge Ratio Purge Ratio (1 ppm limit) (1 ppm limit) (30 ppb limit*) 8.1 × 10 8 8.1 × 10 8 Triethyl amine Triethyl amine 16200 16200 486 7.3 × 10 9 7.3 × 10 9 DMF DMF 36500 36500 1095 * Assumes quantitative conversion of the amine precursor into a nitrosamine, in itself highly unlikely. Linked to control limits for Sartans in Article 31
De-risking Candesartan (AZ) Initially • > 40 batches of API tested – NDMA not detected (LoD 150 ppb) • DMA not detected in Stage 5 (LoD 100 ppb) • Nitrite not detected after Stage 5 Now • Option 4 backed up by testing • >85 batch analyses for NDMA and NDEA (LoD 5 ppb) • >65 batches tested for 5 nitrosamines This work has now been published: Org. Process Res. Dev. Just Accepted Manuscript , doi.org/10.1021/acs.oprd.0c00264
Exploring potential reactivity of nitrosamines
Nitrosamines: structure and overall reactivity Russ. Chem. Rev. 1971 , 40 , 34-50 The chemistry of Amino, Nitroso and Nitro compounds and their derivative 1982, 1151-1223.
Nitrosamine Reduction • Knowledge of reactivity of nitrosamines under the following conditions: • LiAlH 4 Strongest evidence of purge • Zn, Acid (HCl, AcOH) • H 2 , RaNi • SnCl 2 Evidence of purge – but limited quantity • NaBH 4 , Lewis acid • H 2 , Pd/Pt Variable purge – highly dependant on • conditions and/or competition DIBAL
Nitrosamine Reduction LiAlH4 Zn/aqueous acid Fe/aqueous acid H2/metal catalyst NaBH4/Lewis acid 0 20 40 60 80 100 120 140 Number of reported yields for reducing agents o Readily reduced - strong hydrides, zinc or iron in aqueous acid. o Readily reduced by Raney nickel. o Can be reduced by sodium borohydride with the addition of a Lewis acid (e.g. NiCl 2 , TiCl 4 ). o Moderate reactivity with DIBAL. o No evidence of reduction by boranes (i.e. BH 3 ), although C -nitroso compounds are reduced.
Nitrosamine Reduction WO2019236710A1 WO2003106457A1
Nitrosamine Reduction • <100 results with reported yields • Majority use Raney nickel • RT, alcohol solvents and short reaction times • Amine is the main product • Reactivity is catalyst and condition dependent Synthesis 1976 , 548-550
Nitrosamine Reduction: Raney Nickel J. Am. Chem. Soc. 2013 , 135 , 468-473 Synthesis 1976 , 548-550 J. Org. Chem. 1986, 51, 14, 2687-2694
Nitrosamine Reduction: Palladium Tetrahedron 1997 , 38 , 619-620 J. Antibiot. 1993 , 46 , 1716-1719 J. Chem. Soc., Perkin Trans. 1 , 1990 , 3103-3108
Nitrosamine Reduction: Platinum J Med Chem 1984 , 27 , 1710 - 1717 Helv. Chim. Acta 1980 , 63 , 2554-2558
Nitrosamine Oxidation • Oxidations of nitrosamines have limited public data available. • Knowledge of reactivity of nitrosamines under the following conditions: • H 2 O 2 + AcOH/TFA Strongest evidence of purge • H 2 O 2 • KMnO 4 • MnO 2 Available evidence suggests limited purge • Chromium Oxidants • DMP • mCPBA/DMDO • Ozone, oxone, Swern No data available
Nitrosamine Oxidation Majority of evidence of oxidation to nitramines is with peroxide reagents: Synthesis, 1985, 1985, 677-679 US20090286994A1 J. Am. Chem. Soc., 1954, 76, 3468-3470
Nitrosamine Oxidation Limited evidence of nitrosamine oxidation with inorganic reagents: Org. Lett. , 2017, 19, 894-897 Ber. Dtsch. Chem. Ges ., 1901, 34, 1642-1646 Chem Res Tox. , 2000, 13, 72-81 Org. Lett. , 2017, 19, 894-897
Nitrosamine Denitrosation HCl TFA H2SO4 HOAC HCl/CuCl chlorosulfonyl isocyanate HClO4 0 20 40 60 80 100 120 140 160 180 200 Number of examples with reported yields • Normally carried out with aqueous acids (e.g. HCl, TFA, H 2 SO 4 , AcOH, HBr) • Alternative methods have been reported: CuCl/HCl, BF 3 • THF/NaHCO 3 (aq), chlorosulphonyl isocyanate
Nitrosamine Denitrosation Syn. Commun. 2015 , 45 , 2030-2034 Org. Biomol. Chem. 2014 , 12 , 8390-8393 • The equilibrium is dependent on the acid, nitrosamine and temperature. • Hydrolysis normally occurs in aqueous acid at pH < 3 • HCl (0.5 – 5 M) and H 2 SO 4 (50 – 80%) are two most commonly used acids. • HCl and HBr are very efficient as the halide nucleophile can facilitate amine release. • Removal of the amine or NOX from the reaction is necessary for complete reaction. • Typical NOX ‘traps’ are: NaN 3 , HN 3 , urea, sulphamic acid, hydrazine, MeOH, EtOH.
Nitrosamines and Organometallics Grignard reagents: Nitroso nitrogen alkylation followed by α -carbon alkylation with excess Grignard reagent to form trisubstituted hydrazines. Farina PR et al., J. Org. Chem., 1975, 40, 1070-1074
Nitrosamines and Organometallics Organozinc reagents: Grignard reagents: Nitroso nitrogen alkylation followed by α -carbon alkylation with Violent reaction with diethylzinc: excess Grignard reagent to form trisubstituted hydrazines. Lachman A, Am. Chem. J., 1899, 21, 433-446 No reaction with diethylzinc: Farina PR et al., J. Org. Chem., 1975, 40, 1070-1074
Nitrosamines and Organometallics Organolithium reagents: Nitroso nitrogen alkylation, followed by dimerization to form hexahydrotetrazines. Farina PR et al., J. Org. Chem., 1973, 38, 4259-4263 Nitroso nitrogen alkylation, followed by α -carbon alkylation to form trialkylhydrazines. Vazquez AJ et al., Synth. Commun., 2009, 39, 3958-3972
Summary There is limited good quality data in the literature for nitrosamine reactivity – only 4 main transformations. • Reduction is highly dependant on the reductant: • Oxidation is highly dependant on the oxidant: • Hydrogen peroxide Lithium aluminium tetrahydride • Zinc or iron in acid Hydrogen peroxide and acetic acid/trifluoroacetic acid • Hydrogen with Raney nickel • Denitrosation by acid hydrolysis requires relatively high acid concentrations and a trap . • Organometallic addition can occur, but data is limited. • There are significant areas that need further experimental investigation: • Hydrogenation catalyst/conditions • Inorganic oxidising agents
Formation of N -nitroso compounds (NOCs)
Classical Nitrosamine Formation • NOC formation is dominated by N -nitrosation of a NH-containing compound with a nitrosating agent • amine (secondary/tertiary) • (hetero)amide • Carbamate • hydroxylamine • hydrazine Reactive [NO] + carriers: 6 main species • [NO] + precursors: numerous reagents •
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