MedChemComm
Research Article
halogen atoms for the SNAr and that of the nitrile, vinyl and
ethynyl groups for the AdN reactions. We have compiled the
library from 84 electrophilic heterocycles, out of which 27
were synthesized in our laboratory. The pyridine, pyrimidine,
imidazole and pyrazole rings were substituted at three differ-
ent positions; the oxazole and thiazole rings at two positions;
and finally the pyrazine and isoxazole at one available posi-
tion. Notably, in some cases, the 1-substituted compound was
not available; therefore, 2-chlorobenzoxazole, 2-bromo- and
2-vinyl-5-phenyloxazole, 3,5-dimethylisoxazoles and 2-chloro-
benzothiazole were considered (see Tables 1 and S1† for the
chemical structures of the library).
First, the stability and reactivity of the library members
were investigated in a GSH-based assay (Fig. 1) using HPLC-
MS (Fig. 1 IIa) or NMR-based kinetic methods (Fig. 1 IIb).12
We measured the decreasing amount of the electrophilic
fragment up to 72 h in two parallel measurements. The aque-
ous stability of the compounds was characterized by the frag-
reactive, with t1/2IJGSH) > 72 h (shown in Table S1†). Since
the library is intended for use in labelling cysteine nucleo-
philes, we considered two major subsets of the compounds.
The first subset involves compounds with nitrile, vinyl and
ethynyl substituents that react through nucleophilic addi-
tion (AdN). The second set consists of halogenated deriva-
tives that label cysteine in nucleophilic substitution reac-
tions (SNAr). Comparing the six-membered fragments, we
found that halogenated compounds showed weak reactivity
(for A1, A2, A3, B2, C1, C2, C3, D2, E1, F1, F2, G1, t1/2IJGSH)
> 69 h, and B1, B3, D1, D3, F3, G2, G3 were essentially
non-reactive). In contrast, the cyano-, vinyl- and
ethynylpyridines at position 4 (C4 (0.8 h), C5 (0.3 h), C6
(2.4 h), respectively) reacted quickly (Table 1). In the pyrimi-
dine subset equipped with CN or ethynyl groups, position 2
between the two nitrogens gave the shortest half-lives (D4
(2.2 h), D6 (46.8 h) (Table 1)). Among vinylated six-
membered heterocycles, pyridines were the most potent
electrophiles (A5 (1.0 h), C5 (0.3 h), Table 1).
ment half-life calculated from the equation t1/2IJdeg) = ln 2/kdeg
,
where the degradation rate constant for auto-degradation
(kdeg) was calculated by linear regression of the measured
datapoints in the absence of GSH. Thiol reactivity was
assessed by measuring fragment depletion with a large excess
of GSH12 that provided the rate constant kdeg+GSH as the sum
of the thiol-reactivity and the degradation. The GSH reactivity
of the electrophile was then calculated from these two rate
constants as kGSH = kdeg+GSH − kdeg. The GSH half-life (t1/
2IJGSH)) was determined from the kGSH thiol-reactivity rate
constant.
Stability data confirmed that all of the compounds showed
the appropriate stability (>1 h) required for biological testing
(Table S1†).3 Notably, the less stable species were found in
isoxazoles (t1/2IJdeg) < 17 h), while 2-chlorobenzoxazole and
2-chlorobenzothiazole also had t1/2IJdeg) < 24 h.
Taking a closer look at the cyano derivatives, position 4 of
the pyridine (C4, 0.8 h) and position 2 of the pyrimidine (D4,
2.2 h) and the pyrazine (G4, 22.5 h) rings were most reactive
(Table 1). Focusing on the five-membered heterocycles,
among the imidazole derivatives, only 2-iodoimidazole (H3,
6.0 h) showed considerable reactivity, and among pyrazoles,
only 3-ethynyl- (K6, 4.9 h) and 4-ethynylpyrazole (L6, 1.7 h)
were reactive (Table 1). In the case of the oxazole core, the
2-iodo- (N3, 0.1 h), 2-cyano- (N4, 0.5 h) and 4-cyanooxazole
(O4, 1.0) showed remarkable reactivity (Table 1). From the
3,5-dimethylisoxazoles, only the 4-ethynyl derivative (P6, 5.4
h) was reactive (Table 1). Thiazoles were, in particular, the
most reactive heterocycles in the five-membered group. Their
nitrile and vinyl derivatives were most active when located be-
tween the heteroatoms at position 2 (R4 (8.0 h), R5 (2.7 h), re-
spectively, Table 1). In contrast, bromine and ethynyl deriva-
tives (Q2 (63.0 h) and Q6 (53.1 h), respectively) performed
best at position 5 (Table 1).
The results of the GSH reactivity assay revealed that
heterocyclic electrophiles cover a wide range of thiol reactiv-
ity (Tables 1 and S1†). The library contained fragments
reacting under 1 h (C4 (0.8 h), C5 (0.3 h), N3 (0.1 h), N4
(0.5 h), Table 1) to compounds considered practically non-
Next, we analysed the reactivity trends quantitatively using
computed descriptors and experimental (log)t1/2IJGSH) values
Fig. 1 Representation of the (A) HPLC- (IIa) and NMR-based (IIb) thiol-reactivity studies with the (B) corresponding calculations.
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