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Find more information on the Altmetric Attention Score and how the score is calculated. Residual complexity RC involves the impact of subtle but critical structural and biological features on drug lead validation, including unexplained effects related to unidentified impurities. RC commonly plagues drug discovery efforts due to the inherent imperfections of chromatographic separation methods.
The new diketopiperazine, rufomyazine 6 , and the previously known antibiotic, rufomycin 7 , represent a prototypical case of RC that almost resulted in the misassignment of biological activity.
The case exemplifies that impurities well below the natural abundance of 13 C 1.
As a part of lead validation, the dipeptide was synthesized and surprisingly found to be inactive. The initially observed activity was eventually attributed to a very minor contamination 0. This study illustrates the serious implications RC can exert on organic chemistry and drug discovery, and what efforts are vital to improve lead validation and efficiency, especially in NP-related drug discovery programs.
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PDF 1 MB. Abstract High Resolution Image. Historically, chemists have turned to natural sources for novel scaffolds with appealing pharmacological activity. Both misidentification real activity, but incorrect structural identity and misassignment activity assigned to the wrong component of a mixture are common occurrences in natural product NP research and may be attributed to the inherent complexity of naturally derived chemical entities resulting from their metabolomic origin see ref 1 and references therein.
Low abundance constituents that remain throughout the process of fractionation may turn out to be active principles rather than the major, structurally assigned compound. In fact, perfect purity of final pharmaceutical products or active pharmaceutical ingredients APIs is difficult to achieve. Therefore, unless the composition of an API is thoroughly characterized, it is challenging to understand the effects that impurities or other structural variations, including conformers, may have on the biological activity.
However, lead compounds in NP drug discovery programs are seldom characterized with the same scrutiny as pharmaceutical APIs intended for human use. Incorrect chemical characterization leads to the disparity between resources spent on an early phase effort in contrast to the time spent on API validation. This may be due to an exponential relationship between resources spent and the progress achieved.
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The predominance of high-performance liquid chromatography HPLC for purity determination is mainly driven by resource considerations and exacerbates this disparity: representing a relative ratio method, HPLC by default produces outcomes that depend strongly on the chosen detector specific vs universal and evaluation model, including response factors.
In contrast, absolute methods allow definitive purity determination of the target analyte and provide more comprehensive information about the entirety of a sample.
While absolute methods generally require greater resources, there is underutilized potential for even greater time and cost savings down the line when performed at early stages of development. The paclitaxel case also serves as a reminder that highly active minor components, even in low abundance, can significantly affect the outcome of biological activity.
Interestingly, despite a general tendency to under report negative outcomes, the documented cases of misassigned activity can often be attributed to minor contamination arising from highly active components or misidentification of chemical structure.
This study will use the term misassignment when referring to the false assignment of or connection between a specific chemical entity to an observed biological end point. In contrast, the term misidentification is used to designate errors in the process of structure elucidation or dereplication. Recent work in our laboratory led to the isolation of rufomyazine 6 ; Chart 1 , a new diketopiperazine, which after a lengthy path of investigation eventually was recognized as another example of RC that led to the misassignment of activity to an inactive constituent.
Total synthesis and subsequent reisolation eventually led to the conclusion that 6 lacks inhibitory activity against M. It is possible that 6 is a shunt metabolite off the biosynthetic pathway to 7. Supporting this hypothesis, past work done by Schultz et al. This suggests strongly that the required pharmacophore must contain more than just the Trp-Leu portion of 7.
Chart 1. High Resolution Image. The isolation, structure elucidation, and total synthesis of 6 is presented below, in conjunction with a discussion of the global effects of RC in NP drug discovery and methodology to overcome this challenge.
Compound 6 was isolated from the Streptomyces strain, MJU, which was found to be Compound 6 was determined to be a cyclic dipeptide with a molecular formula of C 23 H 31 N 3 O 2 and was recognized as a diketopiperazine by its general NMR characteristics.
The monomeric units were identified as N -methyl- l -leucine N Me- l -Leu and 1- N -isoprenyl— l -tryptophan prenyl— l -Trp. The specific enantiomers come from ECD comparison with the synthetic material see below. Compound 6 shared the same two sequential amino acids with 7 , suggesting that 6 may represent a shunt metabolite from the biosynthetic pathway leading to 7. The signals at Table 1. The 5 1 H NMR signals between 7. The 1 H iterative full spin analysis HiFSA of prenyl— l -Trp Figure 1 indicated the presence of two nearly equally abundant, distinct rotamers ratio: 0.
The five signals between 0.
Additionally, the COSY spectrum clearly connected the entire aliphatic chain of Leu as a continuous spin system. Scheme 1 summarizes the total synthesis of 6.
The intermediate 2 was prepared following the work published by Luzung et al. Electronic circular dichroism ECD analysis of synthetic 6 containing only l -amino acids showed a positive Cotton effect at nm and a negative Cotton effect at nm.
Natural 6 showed identical Cotton effects, indicating that both compounds have the same absolute configuration. Upon initial isolation of 6 , structure elucidation via NMR and HRMS and biological activity assays were run in parallel due to the required 7-day anti- M.
However, the synthetic product, though structurally identical with natural 6 , failed to show any biological activity against M. This led to exploratory measures to determine the source of the activity present in the isolated material.
However, as the sample was less than 1 mg and not crystalline, the potential for minor contamination from a highly potent compound such as 7 had to be considered. The results confirmed the presence of trace amounts primarily 6 and 7.
Based on the MIC values of 7 0.
Thereby, the sample was shown to contain 0. As this is well beyond what typical purity evaluations cover, it raises the broader question about minimum requirements for the RC assessment of biologically active lead samples. However, it is also important to consider purity adjusted bioactivity for any control substances used: for the present rufomycin standard, the purity adjusted MIC was 0.
To perform the purity-adjustment with confidence, the purities of synthetic rufomyazine and the rufomycin standard were assessed by two qNMR absolute and relative and two UHPLC methods Table 2.
Table 2. In addition, to rule out potential for synergistic effects between 6 and 7 , synthetic 6 was assessed biologically in a checkerboard format with 7. The average FIC index value obtained was 0.
The large range of FIC led to a no-effect conclusion. The source of the impurity does not affect the analytical challenge but shall be addressed briefly despite the lack of conclusive evidence. As 6 and 7 were isolated from the same actinomycete strain, same mother fraction, and baseline separated in the same chromatogram see Supporting Information , contamination during sample handling or isolation cannot be ruled out.
An alternative explanation relates to sequential injections during semipreparative chromatography, suggesting that there may have been residual 7 carry-over between injections. This hypothesis was given some preference as 7 analogues tend to elute toward the end of the chromatogram, while 6 elutes early.
It is possible that carry-over of 7 into the collection of 6 was possible. The contamination of 6 by 7 was not anticipated due to drastic difference in retention time and the observations in the NMR spectra.