Quinoxaline-Based Inhibitors of Malarial Protease PfSUB1*

Abstract

Bis(phenylamino)quinoxalines have been identified as a novel class of malarial protease PfSUB1 inhibitors by screening of Malaria Box compounds. The synthesis of analog series and investigation of their inhibitory activity provided preliminary structure-activity relationship to create in silico models for binding of these compounds into the active site of PfSUB1.The alarming spread of drug-resistant malaria has reinforced the search for drugs with a novel mode of action [1][2][3][4]. Inhibition of the malarial protease PfSUB1 (Plasmodium falciparum subtilisin-like serine protease-1) has been shown to be a promising approach to block the propagation of infection by preventing the maturation and egress of merozoites from the red blood cell [5][6][7][8]. Consequently, discovery of small molecule inhibitors for PfSUB1 has become an active field of investigation. However, only a limited number of compounds with activity in the sub-micromolar to micromolar range have been discovered so far [6-10]. Recently, we reported rationally designed peptidic ketoamides as sub-micromolar PfSUB1 inhibitors [11]. In parallel with these studies, we actively searched for non-peptidic inhibitors. For this purpose, we screened the Malaria Box, a collection of 400 compounds which have confirmed activity against the blood stage of Plasmodium falciparum [12]. Screening of this compound library using our previously-described PfSUB1 enzyme assay [11] revealed 2,3-bis(phenylamino)quinoxaline derivative 1a as a PfSUB1 inhibitor (IC 50 10 μM, see scheme and Table 1). Since also several structurally related 2-arylaminoquinoxalines show antimalarial activity in mice [13], as the next step we undertook the synthesis of quinoxaline 1a analogs in order to obtain preliminary structure-activity relationships.

Anti-schistosomal activities of quinoxaline-containing compounds: From hit identification to lead optimisation

“…1 H NMR (500 MHz, DMSO‑ d 6 ) δ 9.52 (s, 2H, 2 x N H ), 8.34–8.18 (m, 3H, 3 x Ar H ), 8.11 (d, J = 8.9 Hz, 1H, Ar H ), 7.87 (m, 2H, 2 x Ar H ), 7.65 (dd, J = 8.8, 6.9 Hz, 3H, 3 x Ar H ); 13 C NMR (125 MHz, DMSO) δ 139.65 (Ar C ), 130.95 (Ar C ), 130.57 (3 x Ar C H), 124.98 (Ar C ), 124.50 (Ar C ), 122.26 (Ar C H), 121.66 (Ar C H), 121.13 (Ar C H), 120.82 (Ar C H), 120.61 (Ar C H), 119.87 (Ar C H); UPLC-MS: Rt (Retention Time): 2.53 min, MS (ESI) + : 496.08 [M+H] + . The spectral data are in accordance with those reported in the literature [ 41 , 42 , 49 ].…”

“…Towards this goal, three compound series (Series A, B and C, clustered based on their chemical similarity) were identified leading to the creation of 21 compounds (re-synthesis of compound 22 and 20 additional analogues, Scheme 1 ). In brief, 2,3-bis(phenylamino)-quinoxalines were obtained from the reaction of 2,3-dichloro-6-nitroquinoxaline with different substituted anilines (resynthesis of compound 22 and synthesis of compound 23–35, Series A) or phenyl-alkyl amines (compound 36 and 37, Series B) as previously described [ 41 , 42 ]. The yields of the reactions reported in Scheme 1 show that the derivatives were obtained in varying amounts reflecting the different reactivity of the anilines and phenyl-alkyl amines used in the reaction.…”

Subtilisin-like Serine Protease 1 (SUB1) as an Emerging Antimalarial Drug Target: Current Achievements in Inhibitor Discovery

“…In silico screening against a PvSUB1 model and assaying of the inhibitory potency for the most promising compounds resulted in a set of five compounds (Figure 15) displaying inhibitory potency at low micromolar concentrations, which provides a good starting point for further development. 33 Compounds 30−32 showed improved activity against PvSUB1 To search for nonpeptidic inhibitors of PfSUB1, the Malaria Box (a collection of 400 compounds with confirmed antimalarial activity) was screened 34 using the PfSUB1 enzyme assay. 15 The screen identified a quinoxaline derivate 35 as a hit compound with an IC 50 of 10 μM (Figure 16).…”

“…To search for nonpeptidic inhibitors of PfSUB1, the Malaria Box (a collection of 400 compounds with confirmed antimalarial activity) was screened using the PfSUB1 enzyme assay . The screen identified a quinoxaline derivate 35 as a hit compound with an IC 50 of 10 μM (Figure ).…”

Peptidic Boronic Acid Plasmodium falciparum SUB1 Inhibitors with Improved Selectivity over Human Proteasome

“…The enzyme is initially stored in a set of merozoite secretory organelles and then discharged into the PV lumen just prior to egress where it cleaves and activates a number of proteins of the PV and merozoite surface. This rapidly leads to explosive rupture of the PV membrane (PVM) and RBC membrane to allow the release of invasive merozoites. − A single orthologue of SUB1 is found in the genomes of all known Plasmodium species, and a critical role of SUB1 for parasite survival has been genetically confirmed through the demonstration that SUB1 gene disruption results in a complete block in merozoite egress in asexual blood stages of the parasite life cycle and the preceding liver stages of infection. − Several small-molecule SUB1 inhibitors have been discovered either by screening of compound libraries or by rational design based on the established substrate specificity and structure of the enzyme. − Recently, we have developed peptidic boronic acids 1 as P. falciparum SUB1 (PfSUB1) inhibitors with low nanomolar potency (Figure ).…”