A.
Keeley
,
P.
Ábrányi-Balogh
and
G. M.
Keserű
*
Medicinal Chemistry Research Group, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok krt 2, H-1117 Budapest, Hungary. E-mail: keseru.gyorgy@ttk.mta.hu
First published on 10th December 2018
A fragment library of electrophilic small heterocycles was characterized through cysteine-reactivity and aqueous stability tests that suggested their potential as covalent warheads. The analysis of theoretical and experimental descriptors revealed correlations between the electronic properties of the heterocyclic cores and their reactivity against GSH that are helpful in identifying suitable fragments for cysteines with specific nucleophilicity. The most important advantage of these fragments is that they show only minimal structural differences from non-electrophilic counterparts. Therefore, they could be used effectively in the design of targeted covalent inhibitors with minimal influence on key non-covalent interactions.
Starting from relevant heterocyclic cores, here we design and characterize an electrophilic fragment library having minimal influence on the potential non-covalent interactions. Based on the well-known electron-withdrawing character of heterocycles,7 our intention was to turn them into fragment electrophiles with the introduction of the smallest available substituents (max. 1–2 atoms). We hypothesized that electron-withdrawing heterocycles activate the small electrophilic substituents and yield warheads suitable for cysteine-targeting covalent inhibitors. Testing this idea, we collected a wide range of five- and six-membered nitrogen-containing heterocycles combined with a selection of small electrophilic warheads. Library members were then subjected to detailed characterization that included the assessment of their cysteine reactivity, specificity and aqueous stability.
Screening electrophilic fragments is now an emerging strategy in both ligand discovery8 and target identification,9 and they can serve as starting points in TCI programs.10 We believe that the library described here might facilitate the development of TCIs by replacing their heterocyclic scaffold with one of our electrophilic heterocycles. This approach would allow the precise positioning of the reactive group toward a catalytic/non-catalytic protein nucleophile in the proximity of the binding site11 while maintaining the key non-covalent interactions.
We planned to investigate the influence of the different heterocycles and the effect of warhead positions by evaluating the fragment's reactivity experimentally. Moreover, we aimed to identify theoretical descriptors supporting the design of new fragments with tailored reactivity. Furthermore, we intended to analyse the reactivity differences between the 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 different positions; the oxazole and thiazole rings at two positions; and finally the pyrazine and isoxazole at one available position. 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-chlorobenzothiazole 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 aqueous stability of the compounds was characterized by the fragment half-life calculated from the equation t1/2(deg) = ln2/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 (t1/2(GSH)) was determined from the kGSH thiol-reactivity rate constant.
Fig. 1 Representation of the (A) HPLC- (IIa) and NMR-based (IIb) thiol-reactivity studies with the (B) corresponding calculations. |
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 (t1/2(deg) < 17 h), while 2-chlorobenzoxazole and 2-chlorobenzothiazole also had t1/2(deg) < 24 h.
The results of the GSH reactivity assay revealed that heterocyclic electrophiles cover a wide range of thiol reactivity (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-reactive, with t1/2(GSH) > 72 h (shown in Table S1†). Since the library is intended for use in labelling cysteine nucleophiles, we considered two major subsets of the compounds. The first subset involves compounds with nitrile, vinyl and ethynyl substituents that react through nucleophilic addition (AdN). The second set consists of halogenated derivatives that label cysteine in nucleophilic substitution reactions (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, t1/2(GSH) > 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 pyrimidine 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).
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 between the heteroatoms at position 2 (R4 (8.0 h), R5 (2.7 h), respectively, Table 1). In contrast, bromine and ethynyl derivatives (Q2 (63.0 h) and Q6 (53.1 h), respectively) performed best at position 5 (Table 1).
Next, we analysed the reactivity trends quantitatively using computed descriptors and experimental (log)t1/2(GSH) values (see Table S2†). The Gaussian09 program package with the B3LYP/6-311++(2d,2p) method and basis set was used to calculate the HOMO (εH) and LUMO energies (εL) and electron distribution on the reacting carbon atom ( and ) at the neutral and also at the −1 charged state, based on the atomic charge distributions in terms of natural population analysis (NPA). From these values, we have calculated the chemical hardness (η = εL − εH), electronic chemical potential (μ = (εL + εH)/2), Parr-index (global electrophilicity, ω = μ2/2η), Fukui-function (frontier function, f+() = ) and local electrophilicity index (ω() = ω·f+()).13 Furthermore, we have computed the transition state enthalpies and Gibbs free energies for the reaction of the chloro- and vinyl-derivatives (modelling SNAr and AdN reactions, respectively) with the MeS− anion as a cysteine surrogate. Inductive sigma constants for the heterocyclic rings were calculated by ACD/Percepta (see Table S2†).
Calculation of the Pearson and Spearman correlation coefficients between each of the descriptors and experimental GSH half-lives revealed no significant correlation when considering the whole library. Taking into account, however, the reaction types between the electrophilic fragments and the targeted nucleophile, we identified some interesting trends. In the case of halogen derivatives reacting in nucleophilic substitutions, we found that the GSH half-life decreases with increasing electronic chemical potential for the halogen set (Fig. 2). Changes in the electronic chemical potential are opposite that of the electron affinity14 and related to the corresponding HOMO energies. This explains why a higher potential is associated with more reactive species with higher εH and with low (log)t1/2(GSH). Notably, the more electrophilic fragments with higher HOMO energies are more reactive towards the nucleophilic cysteine.
Fig. 2 Logarithm of the GSH half-life vs. electronic potential for the halogen set. Standard errors are also shown. |
In the case of ethynyl-substituted fragments reacting in nucleophilic additions, we found that the GSH half-life decreases with increasing atomic charge, which indicates decreasing electron density () for the ethynyl set (Fig. 3). This trend showed that the lower electron density is advantageous for thiol reactivity, since readily ionisable electrophiles and the resulting electron-poor carbon atoms can be considered more reactive towards the thiol nucleophile. This analysis revealed that the prediction of the GSH reactivity requires different descriptors depending on the reaction mechanism. In the case of AdN reactions, the warhead itself does not influence the reactivity, but the local electron distribution on the reacting carbon caused by the electron-withdrawing effect of the aromatic substituent is determining. Moreover, the negative charge is localised on the warhead in the transition state (TS), and the intermediate in AdN reactions implicates the use of a local electronic descriptor. Taking a closer look at SNAr reactions, the aromatic core and the halogen atom should be treated together due to the characteristic leaving group effect. Furthermore, the negative charge is dissipated in the TS. These two factors indicate that a global descriptor is more accurate for the prediction of the reactivity.
Fig. 3 Logarithm of the GSH half-life vs. atomic charge distribution for the ethynyl set. Standard errors are also shown. |
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8md00327k |
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