Controlling a cohort Understanding the risk of nitrosamines within drug substance synthesis - Dr Michael Burns Senior Scientist ...
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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 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, 22-28Utilising 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 31De-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.0c00264Exploring 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:
• LiAlH4
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 competitionNitrosamine 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, 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
WO2003106457A1Nitrosamine Reduction
•Nitrosamine Reduction: Raney Nickel
J. Am. Chem. Soc. 2013, 135, 468-473
Synthesis 1976, 548-550
J. Org. Chem. 1986, 51, 14, 2687-2694Nitrosamine Reduction: Palladium
Tetrahedron 1997, 38, 619-620
J. Antibiot. 1993, 46, 1716-1719
J. Chem. Soc., Perkin Trans. 1, 1990, 3103-3108Nitrosamine Reduction: Platinum
J Med Chem 1984, 27, 1710 - 1717
Helv. Chim. Acta 1980, 63, 2554-2558Nitrosamine Oxidation
• Oxidations of nitrosamines have limited public data available.
• Knowledge of reactivity of nitrosamines under the following conditions:
• H2O2 + AcOH/TFA
• H 2O2 Strongest evidence of purge
• KMnO4
• MnO2
• Chromium Oxidants Available evidence suggests limited purge
• DMP
• mCPBA/DMDO
• Ozone, oxone, Swern No data availableNitrosamine Oxidation
Majority of evidence of oxidation to nitramines is with peroxide reagents:
US20090286994A1 Synthesis, 1985, 1985, 677-679
J. Am. Chem. Soc., 1954, 76, 3468-3470Nitrosamine 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, 72-81Nitrosamine 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 isocyanateNitrosamine 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, 1070-1074Nitrosamines 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, 1070-1074Nitrosamines 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, 3958-3972Summary 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 reagentsNitrosating 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, [HNO2] and Y -
At very acidic media (pH < 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, 2270-2279Non-Classical Nitrosamine Formation
Metal amide nitrosation
Partial reduction Oxidative
of Nitramine
Imidoyl halide
+ nitrate
Nitrosating agentsHighlighting 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 MIsConditions 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
JustificationConditions 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
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