Communications
doi.org/10.1002/cmdc.202100067
ChemMedChem
but showed no inhibition in the Pf cell-based assay at the highest
tested concentration (IC50 >20 μM). Importantly, the cellular assay
occurs over a longer time (72 hours) than the enzymatic assay
followed similar pattern as compound 1 by dimerising mainly
into the predicted NÀ N dimers (Figure 2), m/z: 653.21700 and
m/z: 701.34344, respectively (SI, Figures S49–50), yet cleaner
than compound 1. In comparison, compound 4 showed no
dimerisation, (SI, Figure S51). The substituents seem to define
the overall reactivity of this class of 2-aminothiazoles. For
instance, compound 2 showed in the first screening moderate
activity against PfIspE (IC =191 μM, from a single measure-
(30 min), which may influence the overall stability of the
compound. In addition, we monitored the HRMS chromatographic
profile of freshly dissolved compound 1 in the enzyme assay
buffer (Tris-HCl, pH 7.6) with and without 5% DMSO for 30 min (SI,
Figure S16–17). The presence of DMSO seems to influence the
formation of DP1 (m/z: 380.10260 and 380.10238) and DP2 (m/z:
5
0
ment) that was not reproducible later for a freshly dissolved
365.11554), even within the short time window of 30 min.
batch (PfIspE IC >500 μM), reinforcing the need for careful
5
0
Structure elucidation by NMR spectroscopy showed that the
handling of any related compounds.
mass of DP2 fits in fact to a dimer of 1 (Figure 1). We observed
most often the doubly protonated species with m/z of
In conclusion, we analysed the collected data to understand
what causes the antimalarial activity that evolved from
compound 1 stored in DMSO. The PfIspE activity can be partly
ascribed to dimerised DP2, although it also inhibits PK/LDH. On
the other hand, the oxygenated DP1 may also play a role when
in its active isomeric form. The isolated degradation products
themselves are unstable. They undergo further degradation,
leading to a mixture that may also account for the observed
activities. Additionally, our study underlines the importance of
appropriate storage conditions of DMSO stock solutions of 2-
aminothiazoles. Based on the temperature-dependent studies,
we confirmed that decomposition hardly occurs at À 20°C over
2
+
3
65.11542 [M+2H] more intensely than the charged mass
+
[
M] : 729.22473. With aid of the SFC, we found out that an
isomer of DP2 starts to form over the four days of incubation,
as seen by the appearance of the minor peak, as highlighted in
the green box in Figure 1. NMR measurements revealed its non-
symmetry as one of the characteristic À CH peaks corresponding
to the thiazole position 5 disappeared and a new peak
appeared at 3.96 ppm, integrating for À CH (SI, Table S5). This
2
led us to question how the dimerisation would occur. Based on
the NMR and MS data, we propose that an NÀ N bond formation
occurs between the linker amines, as represented in the
proposed reaction (SI, Scheme S1). NÀ N bonds are generally
unstable, however, rather commonly occur in biological
two months in DMSO. For the future, blocking the 5 position of
the thiazole ring with a fluorine atom could be a feasible
[13]
strategy to reduce the reactivity. However, to avoid decom-
position, working with such a class requires preparation of fresh
stock solutions prior to biological assays. Furthermore, multiple
freeze-thaw cycles should be avoided and alternative solvents
should be considered. With this communication, we wish to
remind the medicinal-chemistry community again that what is
in the test well, might not be the compound one thought.
[11]
complexes. Due to the slightly acidic nature of DMSO, it is
likely that the conjugated pyrrolium species is present and the
imine N, as in tautomer of compound 1, is attacked by the
nucleophilic linker amine forming the NÀ N bond. The charged
pyrrolium may also undergo isomerisation between E and Z
isomers as observed in the SFC conditions, (SI, Figure S38). Z
being slightly more stable than E (+144.7 vs +148.5 kcal/mol),
hampering the exact assignment of the pyrrolium NMR peaks.
For DP2, we could reach a similar enzymatic activity profile as
for the initial decomposed starting points (PfIspE IC =59�
5
0
4
μM), although also inhibiting the auxiliary enzymes (PK/LDH
IC =37�4 μM). The antimalarial activity (PfNF54 IC =2.1�
50
50
0
.2 μM) is also corresponding to an overall increase in activity
as the decomposition occurs (Table 4). The dimerisation via
NÀ N bond formation could also occur at other nitrogen atoms,
supporting the other dimerised masses measured after the first
rp-HPLC (SI, Table S2).
Lastly, we investigated, whether the decomposition of
TM
compound 1 would also occur in Cyrene , similarly dipolar and
[12]
aprotic as DMSO, yet a green solvent. Disappointingly, clear
decomposition occurred even after one day incubation at RT,
although showing different masses to the DMSO samples (SI,
Figure S18). Interestingly, dominating peak (m/z: 476.16010,
TM
potentially with the addition of Cyrene and loss of water)
occurred at 4.68 min, where we previously observed the peak
with DMSO (m/z: 426.12607) and DMSO-d6 (m/z: 432.16534).
This may be ascribed to the general reactivity of compound 1
TM
and Cyrene , which may, nevertheless, still be an interesting
choice for other compounds. Additionally, we screened whether
decomposition would also occur in DMSO at RT for structurally
closely derivatives 2–4. After one day, compounds 2 and 3
Figure 2. Summary of derivatives 2–4 with their proposed dimers of 2 and 3
based on the HRMS measurements (SI, Section 2.9).
ChemMedChem 2021, 16, 1–6
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Ropponen, Henni-Karoliina
