Guanfacine derived Nitrosamines - Are they possible?

Most of the literature on the nitrosation of guanidine derivatives refer to alkyl-substituted guanidines, for which the nitrosation occurs at the alkyl substituted nitrogen., e.g., cimetidine and etintidine. However, there is also information indicating that the nitrosation of dicyandiamide and even guanidine itself is possible, the former having a higher rate constant (Kinetic Study of N-nitrosation of Guanidines, Fernandez et al., J. Phys. Org. Chem., 2011). No structures are indicated.
So far, we have tried to apply the nitrosation of Guanfacine according to the conditions 1 & 2 suggested by EFPIA (Nitrosamines Quality Risk Management Workflows, Aug 2022), detecting 2 impurity peaks by the HPLC-UV. We suspect that these could correspond to nitroso-derivatives.

Would a structure like this plausible:
We will try to do a screening by LC-MS.
Any insight would be appreciated!

As I was recently reminded, there would be no alpha-hydrogen. So, it should not be in the cohort of concern.

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Thank you for the reply! Yes, I am aware that there is no Hydrogen at the alfa Carbons. However, they still may need to be controlled.

Hi, maybe I could give some comments here:

First, I could say that is not an amine but a substitued acetamide and the EFPIA suggestion for nitrosation is related to an amine. Because of the oxygen electron withdrawal properties I do not see realistically possible to a nitroso group to appear in the section of the chemical structure you are looking for with nitrite salts.

  • In all the cases of the mentioned paper this oxygen group is not present (guadinine, etc.)

Second, indeed even if not part of the cohort of concern, evaluation as a possible mutagenic compound is another topic, and goes inside ICH M7.

Nevertheless, if by any instrumental analysis it could de dilucidated that a nitroso group is actually present and stable, that will be the definitive answer.


Thank you for your reply!
My first thought was also that this is a secondary amide and its reactivity should be much lower compared to amines. However, as mentioned above it has been shown that dicyandiamide (cyanoguanidine),
which also includes an electron withdrawing group, generates nitrosamine in mild conditions. Guanidines have a behavior between amines and urea derivatives, therefore the acylated guanidine in Guanfacine may have a different reactivity than a secondary amide.
Regarding that it does not belong to the cohort of concern, in my opinion, it is relevant to supporting a higher AI than 18ng/day, however it is still a nitrosamine

I see your point here, also nitrile is one of the heaviest electron withdrawal groups (or so called deactivation strenght) and still nitrosation happens. However, would be the case that the primary amine that is actually not next to the nitrile group to be the actual nitroso compound being detected?

Or in your case where the imine or primary amine is?

Other comment is that the paper of reference, even though in the introduction it is indicated that guanidines behave similar to amines because of its basicity, the conclusion of the research is quite the opposite that the compounds where the kinetic reaction was quantified behave more similar to amides and ureas.

Nevertheless, your study detected two peaks, as per conservativism of course its elucidation will give the definite answer as well as understanding if are stable.

Finally, I do agree that evaluating this compound at least as per ICH M7 makes sense. Any in-silico evaluation to see its potential mutagenicity could also help.


Thank you for following up!
Indeed, by HPLC_UV there have been 2 impurity peaks detected which do not correspond to degradants obtained during forced degradation studies. This suggests that there are two compounds possible, of which one could have the structure represented in the original posting.

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This is a guanine. What you have here are NITROSAMIDES and not NITROSAMINES. Please be aware of this difference and not open a Pandora’s box that would cause the industry more problems. Nitrosamides are fomed, but they are very different from nitrosamines. They are direct acting mutagens and carcinogens. So a negative Ames (no modification) should tell you if they mutagenic. However, we had a lot of discussion in this forum on this topic. I have also published a white paper, trying to caution industry regarding this difference. Please, please have a look and make a decision.


The clarification is really useful and appreciated!
Indeed, these compounds would belong to the class of nitrosamides not nitrosamines and they do not require metabolic activation, therefore the nonexistence of alfa Hydrogen is also irrelevant.
However, we are still looking if nitrosated compounds or some other degradation products resulted in the reaction with nitrite.

Look at some of the references I have in my paper. Alkyl guanidines form nitroso compounds quite easily. so, you will see them.


Please, share your data with the group once you have run the LCMS samples.

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I will post any relevant data we may obtain

Further improvement of the elution conditions showed 3 impurity peaks, eluting later than Guanfacine. LC-MS seems to confirm the formation of 2 nitroso derivatives, 1st having m/z 275-277 Da and a second 303-305 Da (smaller peak - possibly double nitroso derivative?). Between these two, there was a third peak having m/z 262-264.
Based on the HPLC-UV data, the maximum amount generated was NMT ca. 0.5%, for the larger peak after 24h. The reaction appears to attain an equilibrium at low conversion values.

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