Identification of 2-arylquinazolines with alkyl-polyamine motifs as potent antileishmanial agents: synthesis and biological evaluation studies

Abstract

2-Arylquinazolines with a range of alkyl polyamines as side chain/ring functional motifs at the 4th-position were considered for antileishmanial study with the rationale that related heterocyclic scaffolds and polyamine functionalities are present in drugs, clinical trial agents, natural products and anti-parasitic/leishmanial agents. Synthesis involves construction of the 2-arylquinazolin-4-one ring and deoxyamination via deoxychlorination followed by S_NAr-based amination or a methodology of S_NAr–deoxyamination driven by BOP-mediated hydroxyl-activation. Various alkyl-polyamines important for activities were incorporated. A total of 26 compounds were prepared and screened against Leishmania donovani (Ld) promastigote cells using the MTT assay. Most of the investigated series of compounds showed characteristic leishmanicidal properties. Several compounds showed pronounced leishmanicidal activities (IC₅₀: 5–6.5 μM) with higher efficiency than the antileishmanial drug miltefosine (IC₅₀: 10.5 μM), and relatively less cytotoxicity to macrophage host cells (SI: 9.27–13.5) compared to miltefosine (SI: 3.42). Important pharmacophoric skeletons were identified.

A strategy for scaffold-hybridization of drugs, clinical trial agents, and natural products has led to identification of new chemotypes as antileishmanial agents more effective than the oral-drug miltefosine. Unique pharmacophores have been explored.

Introduction

Leishmaniasis is the second worst neglected tropical disease after malaria. It is caused by more than 20 Leishmania parasite species and transmitted to humans by infected sand fly, Phlebotomus argentepis.¹ Leishmaniasis occurs in three major forms, visceral, cutaneous and mucocutaneous. Visceral leishmaniasis (VL) is the most fatal form. No vaccine is yet available. The currently used antileishmanial drugs are amphotericin B, paromomycin and miltefosine. pentavalent antimonials are not always used due to their widespread resistance. Resistance to drugs in use has also been reported. The common side effects of existing drugs are hepatotoxicity, nephrotoxicity and ototoxicity. Besides, they are required, in general, to be infused daily with a significantly high number of doses. Miltefosine is the only oral drug currently approved, but teratogenicity limits its use.^2–5

As a part of our medicinal chemistry research programme aimed at exploring new chemotype pharmaceutical agents,^6–10 a scaffold hybridization technique of a pool of relevant bioactive heterocycles, drugs, clinical trial agents and natural products as starting molecular templates has been considered in the present work of antileishmanial agent discovery (Fig. 1). Sahu et al. reported that 4-(hetero)-aryl-2-piperazino quinazolines (1) were active against extracellular promastigotes and intracellular amastigotes of L. donovani.¹¹ Agarwal et al. demonstrated a tetrahydrobenzoquinazoline derivative (2) as an antileishmanial compound and identified the importance of the pyridylpiperazinyl group and 4-chlorophenyl ring substitutions.¹² 2,4-Diaminoquinazoline analogs of folate (3) showed high activity against L. major, as reported by Berman et al.¹³ A tryptanthrin derivative (4) exhibits remarkable activity against L. donovani amastigotes.¹⁴ Quinazoline-2,4-diamine (5) was found to be effective against the growth of L. donovani.¹⁵ The antileishmanial potential of a broad range of 4-arylamino-6-nitroquinazoline compounds was explored by Saad et al. The compound with 4-methylsulfanyl substitution (6) was found to be significantly cytotoxic against L. major and more potent than pentamidine.¹⁶ In an investigation of the activity of quinazoline-2,4,6-triamines against Leishmania mexicana, the N⁶-(ferrocenmethyl) derivative (7) showed significant activity against promastigotes and intracellular amastigotes.¹⁷ Licochalcone A (8), a natural product isolated from the roots of Chinese licorice, inhibits both L. major and L. donovani.¹⁸ Romero et al. identified a series of 2-aryl-quinazolin-4(3H)-ones (9) effective against L. mexicana, L. braziliensis and L. amazonensis parasites (EC₅₀ 4–10 μM).¹⁹

Fig. 1. Aminoquinazolines and analogues previously reported as antileishmanial agents.

In addition, various side chain/ring polyamine functional motifs present in different heterocyclic scaffolds were found to be important for exhibiting antileishmanial activities (Fig. 2). A 4-aminoquinoline derivative (10)²⁰ was found to be more active than the antileishmanial drug amphotericin B. Sitamaquine (11),²¹ a quinoline polyamine derivative, is in phase 2 clinical trial for treatment of leishmaniasis. pentamidine (12), a second line antileishmanial drug, is a bis-amidine derivative.²² Based on the structures of the heterocyclic antiparasitic agents (Fig. 1) and the important functional motifs/side chains (Fig. 2), the title class of compounds was considered for investigation of potential antileishmanial activity.

Fig. 2. A) Various side chain/ring polyamine functional motifs important for antileishmanial activities, and B) the rationale for the 4-aminoquinazoline containing side functional amine as a potent antileishmanial agent; structural relevance based on a previously known drug/agent/natural product.

Results and discussion

Chemistry

The title class of compounds, 2-arylquinazolines with functional polyamine substitutions at the 4-position (15–40), was prepared by an approach for construction of quinazolin-4-ones and their amination with relevant amines (Scheme 1). 2-Arylquinazolin-4-ones (13) were synthesized by the reaction of anthranilamides with aromatic aldehydes under aerobic conditions. The reaction proceeds through imine formation, nucleophilic attack with the ortho-tethered carboxamide functionality, and oxidation by aerobic-oxygen.²³ The chlorination of 2-arylquinazolin-4-ones (13) was performed by employing various known base-mediated^24,25 or base-free²⁶ deoxychlorination methods using POCl₃ as a chlorinating agent. Base-free methods were found to be relatively higher yielding. However, multiple untraceable side reactions occurred under all of these conditions and the maximum yield of the desired product obtained was 35%. As per design of potential antileishmanial agents, the amino-functional alkylamine side chain motif would be an important pharmacophore, and therefore, we desired to incorporate various amino-alkylamines in the investigated compounds. However, these poly-amines were found to cause trouble in the experimental operation and resulted in low yield of products. Further, investigations were done with a variation in the amount of POCI₃, reaction time and temperature. The yield was improved to 55% (Table S1†). With the optimized conditions in hand, 2-aryl-substituted 4-chloro-quinazolines (14a–d) were synthesized. The S_NAr-based amination of compounds (14a–d) with relevant polyamines produced the 2-aryl-quinazoline-4-amines with a yield of 70–95% of products (15–26).²⁶

Scheme 1. Synthesis of 2-aryl-quinazoline derivatives with functional amines at the 4-position; i) anthranilamide (1 mmol), aldehyde (1 mmol), DMSO, open air, 140 °C, 6–8 h. ii) POCl₃ (2 mL for 1 mmol of substrate), 60 °C, under Ar, 2–4 h. iii) Amine (1.2 equiv.), ⁱPrOH, 80 °C, 2–5 h. iv) BOP (1.5 equiv.), DBU (1.3 equiv.), amine (1.5 equiv.), MeCN, 30 °C, 3–4 h.

Phosphorus oxychloride is a hazardous chemical. The procedure for deoxygenation of carbonyl with POCl₃ causes harsh and high acidic conditions to substrates. An experiment using it requires stringent anhydrous conditions and is a tedious procedure. Therefore, we conducted an investigation to identify a procedure for amination of 2-arylquinazolin-4-ones avoiding deoxychlorination, which would also reduce the number of reaction steps. Phosphonium-based coupling reagents are frequently used for amide bond formation of ester, peptide synthesis,^27,28 nucleophilic substitution (S_N) reactions,²⁸ and amination-based transformations in heterocyclic motifs.^29–31 These deoxygenative transformations are governed by the formation of a thermodynamically stable phosphorous–oxygen double bond. One of the most effective phosphonium reagents is benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP). The activation of the hydroxyl group of the tautomerizable heterocyclic ring of carboxamide with BOP was reported for the preparation of cyclic amidines.³² Following this amination procedure, we performed the reaction of 2-arylquinazolin-4-ones (13b–f) with various polyamines in the presence of DBU at room temperature. The direct amination reactions proceeded and the corresponding 2-aryl and 4-amino-substituted quinazolines (27–40) were obtained. The yields of the products were found to be low (12–32%), due to plausible reasons that the investigated amine substrates are primary-amines, have an aliphatic-chain-structure, and are multinucleophilic. However, the method provided numerous advantages. The functional motifs that are susceptible to harsh and acidic reaction conditions, but relevant to biological activities via acting as H-bond acceptors or donors and providing a balance of hydrophilicity–lipophilicity, such as methoxy, N-methyl, hydroxyl, amines, and ethers, remained tolerant of the BOP-mediated deoxyamidination. Our focus was to incorporate into the investigated molecular skeletons a broad array of aryls substituted with methoxy/hydroxyl, halogens, and poly-methoxy/hydroxyl functionalities and a diverse range of alkylamine motifs with varied chain lengths and terminal functionalities, such as free-amine, dimethylamine, morpholine, and N-methylpiperizine, which are relevant to potential antileishmanial activity. All of these motifs were successfully assembled in the title class of molecules. All the products were characterised by spectroscopic (¹H, ¹³C-NMR, and IR) and mass-spectrometric methods (Fig. 3).

Fig. 3. Synthesized compounds evaluated for antileishmanial activities (yield obtained in the last reaction step).

Biological evaluation

The synthesized compounds were evaluated for antileishmanial activity against the WT strain of L. donovani using the MTT assay.³³ Miltefosine was used as the standard drug for the assay. The results are presented in Table 1. Dose dependent inhibition was observed. Out of the 26 compounds evaluated for antileishmanial efficacy, 13 compounds (15, 19, 21, 22, 25, 27, 28, 29, 31, 32, 34, 38, and 40) showed higher activities than the standard drug miltefosine.

In vitro anti-leishmanial activity of 2-aryl-4-alkypolyaminoquinazolines with amino-alkylamines.

Compounds	Anti-promastigote activity IC50 (μM) average ± SD	Cytotoxicity IC50 (μM) THP-1 differentiated macrophage	Selective index (SI)
15	6.5 ± 0.71	62.0 ± 6.00	9.53
16	28.5 ± 3.53	ND	ND
17	12.0 ± 0.00	13.0 ± 3.00	1.08
18	13.0 ± 1.41	63.0 ± 1.00	4.84
19	8.0 ± 0.00	40.0 ± 1.00	5.00
20	NI	—	—
21	7.5 ± 0.70	60.0 ± 0.00	8.00
22	6.5 ± 0.70	15.0 ± 5.00	2.30
23	25.0 ± 1.41	64.0 ± 4.00	2.56
24	32.5 ± 0.70	64.0 ± 4.00	1.96
25	9.0 ± 0.00	>100	∼11.10
26	NI	—	—
27	5.0 ± 0.00	57.0 ± 4.40	11.00
28	5.0 ± 0.00	67.5 ± 2.12	13.50
29	6.3 ± 0.35	53.0 ± 1.40	7.28
30	25.0 ± 4.24	56.0 ± 2.80	2.24
31	5.5 ± 0.70	51.0 ± 1.40	9.27
32	6.5 ± 2.12	65.0 ± 1.40	10.00
33	50.0 ± 28.28	71.0 ± 1.40	1.42
34	6.5 ± 0.70	50.0 ± 1.40	7.69
35	29.5 ± 2.12	61.0 ± 1.40	2.06
36	90.0 ± 7.07	54.0 ± 5.70	0.60
37	NI	NI	—
38	5.5 ± 0.70	37.0 ± 1.40	6.72
39	19.0 ± 5.60	33.5 ± 2.10	1.76
40	6.5 ± 2.12	22.5 ± 2.10	3.46
Miltefosinea	10.5 ± 0.70	36.0 ± 2.80	3.42

^aReference drug for leishmaniasis treatment; N.I. – no inhibition was observed up to 100 μM; N.D.: not determined.

All investigated compounds were further subjected to cytotoxicity analysis using the MTT assay against the THP-1 monocyte cell line.³³ THP-1 monocytes were differentiated into macrophages with the help of phorbol 12-myristate 13-acetate (PMA) and treated with different concentrations of the compounds. The selectivity index values were calculated by dividing the IC₅₀ values of cytotoxicity (THP-1 differentiated macrophage) with the IC₅₀ values of anti-promastigote activity. Six compounds (15, 25, 27, 28, 31, and 32) showed more than three-fold higher selectivity index (>9) than the standard drug miltefosine (3.42). Amongst the investigated series of quinazolines with side chain functional polyamine motifs, compounds 27 and 28 displayed significantly higher activity against promastigote cells and selectivity index. These compounds can serve as potent antileishmanial compounds and are important for their further development.

Measurement of mitochondrial ROS generation

Generation of superoxide radicals upon treatment with compound 27 and compound 28 was monitored for 3–48 h using MitoSOX dye. Both the compounds were unable to trigger any significant intracellular levels of superoxide radicals in the promastigote stage of Leishmania after 3 h of exposure. Maximum induction was observed at 24 h of drug exposure followed by a decrease in the mitochondrial ROS level under treatment of both investigated compounds and positive control antimycin A as shown in Fig. 4. The IC₅₀ and 2X IC₅₀ doses of compound 27 showed 50% and 82% increase, respectively, in the mitochondrial ROS level after 24 h of treatment compared to untreated cells. Meanwhile the IC₅₀ and 2X IC₅₀ doses of compound 28 showed 63% and 119% increase in the mitochondrial ROS level after 24 h of treatment compared to untreated cells. Positive control antimycin A shows maximum fluorescence values. After 48 h of treatment, the samples treated with both compounds showed a lower level of mitochondrial ROS, although there is no significant change in 48 h in untreated cells and vehicle control justifying the low cell count due to cell death by compound 27 and compound 28. The data indicates that the investigated compounds 27 and 28 exhibit antileishmanial activity by promoting a significant increase in reactive oxygen levels.

Fig. 4. Mitochondrial ROS generation in Leishmania promastigotes upon treatment with compound 27 and compound 28. The effects of compound 27 (4.A) and compound 28 (4.B) on mitochondrial ROS levels measured by MitoSox Red in promastigotes treated for 3 h, 6 h, 24 h, and 48 h were studied. The figure shows the results in relative fluorescence units (RFU) and fold relative to untreated control cultures. Results shown correspond to mean ± standard deviation (S.D.) of two independent experiments and asterisks represent significant differences related to untreated cultures (***: p < 0.0001 **: p < 0.001 *: p < 0.01).

Effect of selected compounds on recombinant Leishmania donovani trypanothione reductase (Ld-TryR) enzyme

The recombinant Ld-TryR enzyme was expressed and purified using Nickel-NTA affinity chromatography. Protein estimation was done for each eluted fraction using the BCA method. Recombinant Ld-TryR enzyme inhibition was performed for compounds 27 and 28 with increasing concentration (5–100 μM) using 4-chloromercuribenzoic acid (CMB) as a standard inhibitor. No effect of compounds 27 and 28 was observed on the recombinant trypanothione reductase enzyme (Fig. S-1A and B†). This study indicates that the Ld-TryR enzyme is not associated with the mitochondrial ROS generation caused by the investigated compounds 27 and 28.

Structure–activity relationship (SAR) study

The structure–activity relationship (SAR) study of 2-aryl-4-alkypolyaminoquinazolines as antileishmanial agents indicated the unique pharmacophoric importance of motifs (Table 1 and Fig. 4). Dimethylaminopropyl amine and dimethylaminoethyl amine at the 4th position of 2-aryl-quinazolines and the functionalities 4-hydroxyl and 4-methoxy functional groups on the phenyl ring displayed high anti-promastigote activity (IC₅₀: 5–5.5 μM for compounds 27, 28, 31 and 38). The replacement of 4-hydroxyl and 4-methoxy functional groups with 4-chloro and 4-fluoro at the phenyl ring (compounds 15–26; IC₅₀ 6.5–32.5 μM) decreases the activity irrespective of the presence of alkylpolyamine at the 4th position. However, the presence of multiple methoxy and hydroxyl groups on the phenyl ring does not contribute to enhancing the activity. The replacement of hydroxyl by methoxy at the 4th position of phenyl (compound 27vs.31 and compound 28vs.32) increases the activity. Nevertheless, replacement of dimethylaminopropyl amine and dimethylaminoethyl amine at the 4th position of 2-aryl-quinazolines with other alkylpolyamines also decreases the antileishmanial efficacy of the compounds (Fig. 5).

Fig. 5. Structure–activity relationship; important pharmacophoric skeletons: motifs and functional groups (FGs) increase the activity from left to right.

Drug-likeness properties

We evaluated the in silico drug-likeness properties of selected potent bioactive molecules 27 and 28 using SwissADME³⁴ software (Table 2). Physicochemical properties (like lipophilicity, water solubility), pharmacokinetic parameters (like BBB permeation, P-gp substrate), drug-likeness filters (Lipinski,³⁵ Ghose³⁶ and Egan filter³⁷) and medicinal chemistry parameters (PAINS,³⁸ Brenk³⁹) were calculated. Also, Bioavailability Radar,⁴⁰ which provides a valuable insight about the oral bioavailability of the compounds, was also evaluated (Fig. 6).

Physicochemical and oral bioavailability-relevant properties of molecules 27 and 28.

Compounds	No. of H-bond acceptors and donors	No. of rotatable bonds	pKαa	TPSA [Å2]	Lipophilicity (log Po/w)		Water solubility (log S)		P-gp substrate	GI abs.	Lipinski	PAINS
					M Log P	SILICOS-IT	ESOL	SILICOS-IT
27	4 and 1	6	8.59	50.28	2.64	3.06	−4.36	−6.92	No	High	0 violation	0 alert
28	4 and 1	7	9.08	50.28	2.86	3.88	−4.58	−7.32	No	High	0 violation	0 alert
Acceptance range	Both ≤ 10 and 5 resp.	≤9	2–12*	≤140b	≤4.15	≤5	−6 < moderately < −4	−10 < poorly < −6	No	High	MW ≤ 500	0
											M Log P ≤ 4.15
											N or O ≤ 10
											NH or OH ≤ 5

^a pK_α is calculated by SimulationPlus version 9.8.2000.

^bVeber filter.⁴²

Fig. 6. Bioavailability radar drug-likeness of molecules 27 and 28.

The hexagonal pink area indicates the optimal range of each descriptor's flexibility – not more than 9 bonds; saturation – not less than 0.25; solubility – not more than 6; topological polar surface area⁴¹ (TPSA) – between 20 and 130 Ų; size – molecular weight between 150 and 500 g mol⁻¹; lipophilicity – XLOGP3 between −0.7 and +5.0. Here both molecules 27 and 28 fall within the hexagonal pink area and possess favourable oral bioavailability properties.

The topological polar surface area (TPSA) is a useful descriptor for crossing biological barriers like the GI track and brain. The SwissADME web tool gives access to five lipophilicity predictive models. Among them M Log P,⁴³ an archetype of topological method relying on a linear relationship with 13 molecular descriptors, is important. SILICOS-IT is a hybrid method relying on 27 fragments and 7 topological descriptors where ESOL⁴⁴ is a water solubility prediction model that shows linear correlation (R² = 0.69) between predicted and experimental values. Pan Assay Interference Structures (PAINS) reported by Baell et al. are molecules containing substructures that show false positive biological output by acting on irregular protein targets.⁴⁵ The present investigated molecules 27 and 28 qualify all these in silico drug-likeness and oral bioavailability parameters.

Conclusion

Medicinal chemistry-based investigations of 2-arylquinazolines with a range of alkyl polyamines as side chain/ring functional motifs at the 4th-position have afforded identification of a number of new compounds as significantly potent antileishmanial agents. Rationale design considering the important heterocyclic molecules and various alkyl polyamine motifs present in the drugs, clinical trial agents, and natural products has been found to be important. In the preparation of the investigated series of compounds, the deoxyamination of 2-arylquinazolinones via a direct S_NAr–deoxyamination driven by BOP-mediated activation was more convenient than the method of deoxychlorination–amination. A total of 26 compounds were prepared with incorporation of a range of substituted aryls and alkyl-polyamines important for activities. The MTT assay indicated that most of the investigated compounds showed characteristic leishmanicidal properties against Leishmania donovani (Ld) promastigote cells. Thirteen compounds showed antipromastigote activities (IC₅₀ 5–9.0 μM) with higher efficiency compared to the antileishmanial drug, miltefosine (IC₅₀ 10.5 μM). They were also found to be relatively less cytotoxic to macrophage host cells. The selectivity indexes of six compounds (15, 25, 27, 28, 31, and 32) were found to be 3-fold higher than that of miltefosine. Amongst the investigated series of quinazolines with side chain functional polyamine motifs, compounds 27 and 28 were identified as the most active (IC₅₀ 5 μM and selectivity index 11–13.5) compared to miltefosine (IC₅₀ 10.5 μM, SI: 3.42). Compounds 27 and 28 showed a significant increase in mitochondrial reactive oxygen levels. However, no effect of these compounds was observed on the oxidative stress-linked enzyme trypanothione reductase. The structure–activity relationship study indicated the previously-unknown pharmacophoric importance of side chain alkylpolyamines at the 4th position and 4-hydroxy and 4-methoxy phenyl at the 2nd position. The in silico study indicates that the investigated class of molecules possesses drug-likeness (physicochemical and oral bioavailability) properties. The present study of the identified activity profiles of the investigated molecular motifs is important in terms of enriching the pool of new antileishmanial agents and further investigations.

Author contributions

The manuscript was written through the contributions of all the authors. All the authors have given approval to the final version of the manuscript.

Conflicts of interest

There are no conflicts to declare.