Development of Potent 3-Br-isoxazoline-Based Antimalarial and Antileishmanial Compounds

Abstract

Starting from the structure of previously reported 3-Br-isoxazoline-based covalent inhibitors of P. falciparum glyceraldehyde 3-phosphate dehydrogenase, and with the intent to improve their metabolic stability and antimalarial activity, we designed and synthesized a series of simplified analogues that are characterized by the insertion of the oxadiazole ring as a bioisosteric replacement for the metabolically labile ester/amide function. We then further replaced the oxadiazole ring with a series of five-membered heterocycles and finally combined the most promising structural features. All the new derivatives were tested in vitro for antimalarial as well as antileishmanial activity. We identified two very promising new lead compounds, endowed with submicromolar antileishmanial activity and nanomolar antiplasmodial activity, respectively, and a very high selectivity index with respect to mammalian cells.

3-Br-isoxazoline-based derivatives endowed with antimalarial activity were previously described by our group and their biological properties were correlated to their ability to covalently and irreversibly inhibit cysteine-containing enzymes, in particular P. falciparum glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH), a key glycolytic enzyme playing a crucial role in parasite metabolism.¹ The mechanism underlying covalent inhibition is the nucleophilic attack of the catalytic cysteine residue on the Br-substituted C-3 of the isoxazoline ring.¹⁻³

The most active compounds (i.e., compounds 2–4, Figure 1) were all characterized by the presence of an ester or an amide function in their structure, as they were designed elaborating on the structure of the amino acid 3-Br-acivicin (compound 1, Figure 1), whose antimalarial activity was also reported.²

Figure 1.

Structure of model compounds.

As is well-known, esters and amides may represent an Achille’s heel for biologically active compounds, due to their metabolic vulnerability. Notably, in the case of compounds 2–4, a metabolic conversion into the corresponding carboxylic acid derivative would produce 3-Br-acivicin, which may lead to several off-target interactions, due to the reported ability of this amino acid to inhibit enzymes of the glutamino-amido transferase family, because of its structural similarity with l-glutamine.⁴ Therefore, we sought to increase the metabolic stability of these compounds through bioisosteric replacement of the ester/amide function.

Oxadiazoles are five-membered heteroaromatic rings frequently used in drug-like molecules as bioisosteric replacements for ester and amide functionalities.⁵ On this ground, we first designed a new compound obtained by simply replacing the ester/amide group of compounds 3 and 4 with a 1,2,4-oxadiazole moiety, i.e., compound 5, and then moved to the structurally simplified analogue 6a, where the oxadiazole ring was directly linked to the 3-Br-isoxazoline ring and the amino group was eliminated (Figure 2). Compound 5 turned out to be biologically inactive, whereas compound 6a displayed a moderate antiplasmodial activity (Table 1). From these data, we initiated an investigation of the SARs around this new scaffold, with the aim of increasing its antiparasitic activity. At first, we studied the effect of different functionalization on the aromatic ring appended at the 3 position, and then we replaced the oxadiazole ring with several different five-membered heterocyclic scaffolds. Finally, on the basis of the results of the phenotypic assays and the SAR analysis, we combined the best structural features all in one (Figure 2).

Figure 2.

Med-chem strategies used for the design of new compounds starting from lead compounds 3 and 4.

Table 1. In Vitro Activity of 3-Br-isoxazoline-Based Inhibitors against Plasmodium falciparum (D10 and W2 strains) and Leishmania spp. (L. infantum and L. tropica) Promastigotes.

compd	P. falciparum D10 IC50 (μM)a	P. falciparum W2 IC50 (μM)a	L. infantum IC50 (μM)b	L. tropica IC50 (μM)b
3	0.37 ± 0.12	0.26 ± 0.05	n.d.	n.d.
4	0.36 ± 0.11	0.48 ± 0.18	n.d.	n.d.
5	inactivec	inactivec	n.d.	n.d.
6a	52.7 ± 14.4	64.8 ± 9.5	17.3 ± 7.4	43.0 ± 22.6
6b	52.7 ± 14.2	46.0 ± 3.5	26.2 ± 13.1	24.9 ± 8.26
6c	58.3 ± 8.1	48.1 ± 0.1	27.3 ± 12.3	32.6 ± 12.7
6d	inactivec	inactivec	9.0 ± 2.7	10.9 ± 2.8
6e	31.0 ± 6.9	34.0 ± 14.8	8.7 ± 4.5	6.9 ± 2.6
6f	3.1 ± 0.1	3.5 ± 0.4	0.29 ± 0.11	0.31 ± 0.03
6g	34.4 ± 5.8	42.5 ± 6.0	12.0 ± 2.0	14.7 ± 8.1
6h	29.8 ± 4.5	32.7 ± 2.8	25.6 ± 6.2	39.4 ± 11.8
6i	27.8 ± 1.1	27.2 ± 1.5	10.5 ± 4.4	5.3 ± 2.8
6j	36.9 ± 5.0	45.3 ± 4.8	inactivec	inactivec
6k	45.9 ± 7.5	40.4 ± 13.0	inactivec	inactivec
6l	45.7 ± 12.2	39.5 ± 17.0	28.8 ± 5.0	33.8 ± 24.2
6m	39.0 ± 16.3	32.5 ± 12.9	14.7 ± 6.16	25.6 ± 11.7
7	16.7 ± 8.3	15.1 ± 7.6	inactivec	inactivec
8	35.3 ± 15.9	43.0 ± 7.1	inactivec	inactivec
9	32.2 ± 14.7	39.3 ± 11.8	inactivec	50.7 ± 20.1
10	22.9 ± 9.6	41.3 ± 8.8	60.3 ± 6.2	inactivec
11	45.8 ± 3.9	39.7 ± 3.9	28.8 ± 5.0	33.8 ± 24.2
12	0.035 ± 0.014	0.058 ± 0.014	3.5 ± 1.2	7.5 ± 3.4
CQ	0.028 ± 0.004	0.406 ± 0.113	n.d.	n.d.
AMB	n.d.	n.d.	0.11 ± 0.02	0.12 ± 0.02

^apLDH method; data are the mean of three different experiments ± SD; D10: CQ-susceptible Pf strain; W2: CQ-resistant Pf strain; n.d.: not determined.

^bMTT assay; data are the mean of three different experiments ± SD; n.d.: not determined.

^cNo activity observed at the highest concentration tested (60 μM).

Considering that GAPDH, the putative target of this series of compounds, is not only a key enzyme in P. falciparum metabolism but plays also a pivotal role in the glycolytic pathway of kinetoplastida protozoan parasites,⁶ we decided to broaden our biological investigation, evaluating their activity both against P. falciparum and two species of Leishmania, i.e., L. tropica and L. infantum.

The synthesis of the 1,2,4-oxadiazole derivatives 6 started from the appropriate nitriles (14a–m), which were commercially available or, in the case of nitriles 14i and 14k, were synthesized from their corresponding aldehydes (13i, 13k) using a one-pot procedure that involves the transformation of the aldehyde group into an aldoxime, acetylation, and loss of acetic acid to afford the nitrile⁷ (Scheme 1).

Scheme 1. Synthesis of Compounds 5 and 6a–m.

Reagents and conditions: (a) NH₂OH*HCl, pyridine, Ac₂O, reflux, 3 h; (b) NH₂OH*HCl, Na₂CO₃, EtOH/H₂O (10:1 v/v), reflux, 4–24 h; (c) acryloyl chloride, dry THF, 1–3 h; (d) 1 M TBAF in dry THF, 30–60 min; (e) DBF, NaHCO₃, EtOAc, overnight; (f) Boc₂O, I₂, overnight; (g) MOMCl, DIPEA, dry DCM, 2 h; (h) 30% TFA/DCM, 4 h; (i) BiCl₃, CH₃CN, H₂O, 50 °C; (j) benzamidoxime, PyBOP, DIPEA, dry DCM, overnight.

Before the following step, nitrile 14l had to be protected as N-Boc (14n) using di-tert-butyldicarbonate (Boc₂O) in the presence of a catalytic amount of molecular iodine under solvent-free conditions,⁸ whereas nitrile 14m was converted into the corresponding O-MOM protected derivative (14o) by reacting it with chloromethyl methyl ether (MOMCl) in the presence of diisopropylethylamine (DIPEA) (Scheme 1). Nitriles 14 were converted into amidoximes 15, by refluxing them with hydroxylamine hydrochloride and sodium carbonate in EtOH/water. The so obtained amidoximes were chemoselectively O-acylated with acryloyl chloride obtaining O-acrylamidoximes 16. The following cyclization reaction represents the key step of this synthetic route. A systematic analysis of the cyclization conditions was reported in the literature by Baykov and co-workers,⁹ who highlighted the use of the superbase system KOH/DMSO as the more efficient method for a wide range of heat-labile intermediates. However, in a recent publication,¹⁰ the same research group used this method to prepare a number of variously decorated vinyl-1,2,4-oxadiazoles and obtained compound 17a in only an 11% yield, explaining this outcome with the generation of a base-initiated anionic polymerization subproduct due to the Michael acceptor behavior. Therefore, we decided to explore different reaction conditions, using compound 16a as a model substrate and four different bases (DBU, Py, TBAF,¹¹ K₂CO₃) at three different temperatures (rt, 60 °C, 100 °C) in dry THF. In our hands, the best results in terms of yield and purity were achieved using a catalytic amount of TBAF (0.2 equiv) at room temperature, affording oxadiazole 17a in only 1 h and 97% yield. Accordingly, all the desired 3-substituted-5-vinyl-1,2,4-oxadiazoles 17 were prepared following this optimized procedure.

The vinyl oxadiazoles 17a–k, 17n, and 17o were then submitted to 1,3-dipolar cycloaddition reaction with bromonitrile oxide, generated in situ from its stable precursor dibromoformaldoxime (DBF), in the presence of solid NaHCO₃ as heterogeneous base and ethyl acetate as a solvent, according to a well-established literature procedure for the synthesis of 3-bromo-isoxazoline derivatives.¹²

The cycloaddition reaction afforded the desired 3-Br-isoxazoline compounds 6a–k, 6n, and 6o (Scheme 1).

At this stage, 6n was deprotected with a 30% solution of TFA in DCM to give the amine derivative 6l, whereas O-MOM derivative 6o was converted into the corresponding phenol 6m with BiCl₃ as a Lewis acid catalyst.¹³

The same optimized procedure for the 1,2,4-oxadiazole synthesis was used to prepare compound 5. In this case, the oxadiazole ring formation was performed after the 1,3-dipolar cycloaddition step, using the already available synthetic intermediate 18, previously described by us,² which was reacted with benzamidoxime, using benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and DIPEA as coupling agents to give the N-acyloxybenzimidamide derivative 19. Cyclization with catalytic TBAF in THF at room temperature afforded the 1,2,4-oxadiazole 20. Finally, treatment with a 30% solution of TFA in DCM afforded the final compound 5, which was obtained as a free amine after basic aqueous work up (Scheme 1).

The second aim of the present SAR investigation was the replacement of the 1,2,4-oxadiazole ring with different heterocyclic scaffolds, including 1,3,4-oxadiazole, 1,2,4-triazole, oxazole, 1,3,4-thiadiazole, and thiazole, keeping the rest of the molecule unmodified, as in model compound 6a.

Compounds 7–11 were prepared through 1,3-dipolar cycloaddition of bromonitrile oxide to alkenes 21, 23, and 24–26, containing the different heterocyclic scaffolds, using the procedure already described above (Scheme 2).

Scheme 2. Synthesis of Compounds 7–12.

Reagents and conditions: (a) DBF, NaHCO₃, EtOAc, overnight; (b) acryloyl chloride, K₂CO₃, Et₂O, overnight; (c) N₂H₄*H₂O, MeOH, 2 h; (d) potassium trifluorovinylborate, Pd(PPh₃)₄, 2 M aq. Na₂CO₃, EtOH, toluene, reflux, 2 h; (e) acrylic acid, EDC*HCl, DCM, overnight; (f) hexachloroethane, PPh₃, DIPEA, MeCN, 2 h.

The desired alkenes were prepared as follows. Compound 21 was prepared starting from commercially available methyl benzoate, following the synthetic route reported by Milinkevich and co-workers.¹⁴ The 1,2,4-triazole 23 was prepared employing the synthetic route developed by Azzouni et al.¹⁵ with some minor modifications: methylbenzimidate 22, in turn obtained from benzonitrile according to a published procedure,¹⁶ was treated with acryloyl chloride in the presence of solid K₂CO₃ to give the intermediate methyl N-acryloylbenzimidate, which was then cyclized with hydrazine to afford 23. Oxazole 24 was prepared as reported in the literature, starting from commercially available 2-Br-acetophenone.¹⁷ Alkenes 25 and 26 were obtained through a Suzuki cross-coupling by reacting the commercially available brominated precursors with potassium vinyltrifluoroborate and Pd-tetrakis. (Scheme 2).

Finally, after having submitted both series of compounds 6a–m and 7–11 to biological evaluation, as discussed in detail below, we combined the structural features of the more active derivative of each series, i.e., 6f and 7 (Table 1) in the same derivative, thus designing compound 12 (Figure 2), which was synthesized as reported in Scheme 2. 4-tert-Butylbenzohydrazide 27, prepared according to a literature procedure,¹⁸ was acylated with acrylic acid in the presence of EDC*HCl to afford intermediate 28, which was then cyclodehydrated with hexachloroethane/PPh₃ and DIPEA, to give 29, following the same procedure used to prepare compound 21.¹⁴ Alkene 29 was finally submitted to the 1,3-dipolar cycloaddition step to produce 12.

The phenotypic assays were conducted on D10 (chloroquine-sensitive) and W2 (chloroquine-resistant) strains of P. falciparum, using the parasite lactate dehydrogenase (pLDH) method and chloroquine (CQ) as control,^19,20 and on L. tropica and L. infantum promastigotes, using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method and amphotericin-B (AMB) as a control.^21,22

The activity of the tested compounds is reported in Table 1. The antiplasmodial activity was compared to that previously reported for our model compounds 3 and 4.¹

When the ester or amide function of 3 and 4 was bioisosterically replaced with the 1,2,4-oxadiazole group (5), quite surprisingly, the antiplasmodial activity was completely lost. Conversely, the activity was restored in simplified derivatives, where the amino group was eliminated and the oxadiazole ring was directly linked to the 3-Br-isoxazoline moiety, although this first series of compounds, i.e., 6a–m, displayed only a modest antiplasmodial activity. The IC₅₀s were in the midmicromolar range, with the only exception of compound 6f, which exhibited an IC₅₀ of 3.1 μM against D10 strain and 3.5 μM against W2 strain.

As a general trend, all compounds were 2–10 times more potent against Leishmania parasites, compared to P. falciparum strains. Compound 6f confirmed to be the most active compound, reaching a submicromolar activity against promastigotes of both Leishmania species (IC₅₀ = 0.29 μM against L. infantum and IC₅₀ = 0.31 μM against L. tropica).

In the second set of derivatives 7–11, we replaced the 1,2,4-oxadiazole ring with different pentatomic heterocyclic scaffolds, while keeping the rest of the molecule constant as in compound 6a. However, all the modifications generally led to a modest or negligible increase in the biological activity. The most active derivative of this second set was the 1,3,4-oxadiazole derivative 7, displaying a 3–4 times increase in the antiplasmodial activity if compared to its analogue 6a (IC₅₀ 16.7 μM vs 52.7 μM on D10 strain and 15.1 vs 64.8 μM on W2 strain), although it was inactive against Leishmania parasites.

As a final effort, we combined the structural features of 6f, the best performing derivative of the first set, and those of compound 7, the best of the second set. This structural modification resulted in compound 12, which showed a surprisingly potent antiplasmodial activity. Indeed, this compound displayed an IC₅₀ against D10 strain (CQ-sensible P. falciparum) of 0.035 μM, comparable to the one of the reference compound chloroquine (IC₅₀ = 0.028 μM). Moreover, contrarily to CQ, compound 12 was highly active (IC₅₀ = 0.058 μM) also against the CQ-resistant P. falciparum strain (W2). Interestingly, it also proved to be moderately active against stage IV–V gametocytes from the 3D7 P. falciparum strain, although with a significantly lower potency (IC₅₀ 10.4 ± 4.8 μM).

The activity of 12 against the promastigotes of two Leishmania species was in the low micromolar range (IC₅₀ = 3.5 μM against L. infantum and IC₅₀ = 7.5 μM against L. tropica).

The two more interesting compounds identified in the present study, 6f and 12, were then tested against the BMDM murine macrophage cell line to assess the selectivity indices for parasites versus mammalian cells (Table 2). Compound 6f displayed a selectivity index of 11–12 for P. falciparum vs murine macrophages and >100 for both Leishmania spp. vs murine macrophages.

Table 2. Cytotoxicity of 6f and 12 against Murine BMDM Cells and Selectivity Indices for Parasites vs Murine Cells.

compd	BMDM (IC50 μM)	S.I.aP. falciparum D10/BMDM	S.I. P. falciparum W2/BMDM	S.I. L. infantum/BMDM	S.I. L. tropica/BMDM
6f	37.3 ± 1.5	12	11	129	120
12	40.0 ± 1.1	1143	690	11	5

^aSelectivity index (S.I.) was calculated based on the IC₅₀s against the parasites reported in Table 1.

The latter observation is crucial for the treatment of Leishmania amastigotes, which normally reside and grow inside macrophages, while sparing normal cells.

Compound 12 exhibits a S.I. > 1000 for D10 and >600 for W2 P. falciparum strains vs murine macrophages and only >5–10 for Leishmania spp. vs murine macrophages. Comparable results in terms of selectivity indices were obtained testing compound 12 against human endothelial HMEC cells (data not shown).

Thus, in the present work, starting from the structure of previously reported compounds and using some classical medicinal chemistry strategies such as bioisosterism, structural simplification, and SAR studies, we discovered two very promising antiparasitic agents with potent antiproliferative activity against two different protozoan parasites, P. falciparum and two species of Leishmania. Compared to the initial model compounds 3 and 4, the new derivatives 6f and 12 have completely lost the structural resemblance with the amino acid 3-Br-acivicin, and thus their activity cannot be in anyway led back to their ability to mimic l-glutamine. In view of a future administration in vivo, the toxicity issues deriving from a possible metabolic conversion into 3-Br-acivicin and related metabolites are definitively ruled out. Moreover, the lipophilicity is significantly improved, suggesting better membrane permeability properties compared to the initial compounds; in fact, the calculated Log D_7,4 values are 1.41 for compound 3, 0.76 for compound 4, 3.64 for compound 6f, and 3.35 for compound 12.²³ These new compounds can be prepared through simple chemical steps starting from readily available commercial synthons.

The putative target, on the basis of previous studies on analogue derivatives, is GAPDH, although a target-identification approach will be pursued in the near future to confirm the engagement of GAPDH and identify other possible targets that may covalently bind to the 3-Br-isoxazoline nucleus of 6f and 12.

Interestingly, on passing from the 1,2,4 oxadiazole of compound 6f to the 1,3,4 oxadiazole of 12, we observed a decrease in the antileishmanial activity and a pronounced increase of the antiplasmodial activity. This may underline subtle differences in the binding pocket of GAPDH in the two different parasites or the contribution of a different target.

Compound 6f has a low micromolar potency as antiplasmodial agent and a submicromolar antileishmanial activity, whereas 12 exhibits low micromolar activity against Leishmania spp. and nanomolar potency against P. falciparum. Considering also the very good selectivity of 6f for Leishmania vs murine cells and the even higher selectivity of 12 for P. falciparum vs murine cells, we can conclude that we have identified two very promising lead compounds that deserve further biological investigations as potential antileishmanial (6f) and antimalarial (12) compounds.

Glossary

Abbreviations

AMP

amphotericin-B

BMDM

bone marrow-derived macrophages

Boc

tert-butyloxycarbonyl

CQ

chloroquine

DBF

dibromoformaldoxime

DBU

1,8-diazabicyclo [5.4.0] undec-7-ene

DIPEA

N,N-diisopropylethylamine

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

HMEC

human microvascular endothelial cells

MOM

methoxymethyl

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

pLDH

parasite lactate dehydrogenase

Py

pyridine

PyBOP

benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate

SAR

structure–activity relationship

TBAF

tetra-n-butylammonium fluoride

TFA

trifluoroacetic acid

THF

tetrahydrofuran

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00354.

• Material and methods used in this work, including reagents, synthetic procedures, compound characterization and biological assays (PDF)

Author Present Address

^□ Philochem AG, Libernstrasse 3, CH-8112 Otelfingen, Switzerland

Author Contributions

^† A.G. and A.Z. contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

This research work was supported by funding to D.T., P.C., and N.B. from the Italian MIUR, PRIN2015, project 2015.4JRJPP, and by funding to D.T. from the Italian Ministries of Health and of Foreign Affairs/International Cooperation (MAECI), ISARP PGR00948.

The authors declare no competing financial interest.