Controlling a cohort Understanding the risk of nitrosamines within drug substance synthesis

Dr Michael Burns Senior Scientist [email protected] 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 NaNO2 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 Purge Ratio Purge Ratio (1 ppm limit) (30 ppb limit*) Triethyl amine 8.1 × 108 16200 486 DMF 7.3 × 109 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)

• H2, RaNi

• SnCl2 Evidence of purge – but limited quantity

• NaBH4, Lewis acid

• H2, Pd/Pt Variable purge – highly dependant on • DIBAL conditions and/or competition 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. NiCl2, TiCl4). o Moderate reactivity with DIBAL.

o No evidence of reduction by boranes (i.e. BH3), 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:

• H2O2 + AcOH/TFA Strongest evidence of purge • H2O2

• KMnO4 • MnO2 • Chromium Oxidants Available evidence suggests limited purge • DMP • mCPBA/DMDO

• Ozone, oxone, Swern No data available Nitrosamine Oxidation

Majority of evidence of oxidation to nitramines is with peroxide reagents:

US20090286994A1 Synthesis, 1985, 1985, 677-679

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

Org. Lett., 2017, 19, 894-897 Chem Res Tox., 2000, 13, 72-81 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, H2SO4, AcOH, HBr)

• Alternative methods have been reported: CuCl/HCl, BF3•THF/NaHCO3 (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 H2SO4 (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: NaN3, HN3, 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

Grignard reagents: Organozinc 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: • Lithium aluminium tetrahydride Hydrogen peroxide • 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 Nitrosating agents. NaNO2

• N-Nitrosation by NaNO2 + aqueous acid

- In the absence of a nucleophile, Y = NO2 • Most used and reported method • Nitrosating agent nature depends on pH, [HNO ] and Y - At very acidic media (pH < 2): 2 • Optimum pH depends on amine basicity

• N-Nitrosation by NaNO2 + carbonyl compound + aqueous media

• The reaction rates vary with steric accessibility to the nitrogen atom. - • NO2 /RCHO limited to very electrophilic aldehydes - + • Much less efficient than NO2 /H (aq) Nitrosating agents. NaNO2

• Effective [NO+] nitrosating agents • Heterogenous process • Applied to secondary/tertiary amines, amides, etc.

Synth. Commun. 1999, 29, 905–910 Synth. Commun. 2010, 40, 654-660

Synthesis 2006, 2371-2375 J. Chem. Research 2003, 626-627

Synth. Commun. 2019, 49, 2270-2279 Non-Classical Nitrosamine Formation

Metal amide nitrosation

Partial reduction Oxidative of Nitramine

Imidoyl halide + nitrate

Nitrosating agents Highlighting generation of impurities

Mirabilis 3

• Conversion of an impurity type into a new impurity is flagged within the knowledge base.

• The user can then decide whether to include the resulting impurity as a side/by-product

• No warnings possible for impurities which are created in a process, unless it is transformation of an existing impurity. • E.g. Nitrite and secondary amine

Mirabilis 4

• Understanding of reactions will focus more on the conditions present within a reaction. • This allows for interpretation of chemical combinations resulting in formation of MIs Conditions identified • Primary aromatic amine • Secondary aromatic amine • Aromatic CH • Nitrite • Dilute mineral acid • Transition metal salt Warning Secondary amines are known to generate nitrosamines under acidic conditions when in the presence of a source of nitrite [Ref]

 Risk reviewed

Justification Conditions identified • Primary aromatic amine • Secondary aromatic amine • Aromatic CH • Nitrite • Dilute mineral acid • Transition metal salt Conditions identified • Primary aromatic amine • Secondary aromatic amine • Aromatic CH • Nitrite • Dilute mineral acid • Transition metal salt Summary

• Purge arguments represent a simple yet effective way to both determine the risk of formation and demonstrate their control in line with ICH M7

• There are 4 major mechanisms of reactivity purge: • Reduction (LiAlH4, Zn/Fe in acid, Raney Nickel hydrogenation)

• Oxidation (H2O2 and AcOH/TFA) • Denitrosation (Acid and trap) • Organometallic addition

• Mechanisms of nitrosamine formation exist beyond the scenario of amine + nitrite, and must also be considered within a risk assessment • A review article on nitrosamine formation has been submitted to OPRD Any questions?

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