Synthesis, Antileishmanial Activity and Spin Labeling EPR Studies of Novel β-Carboline-Oxazoline and β-Carboline-Dihydrooxazine Derivatives

Abstract

A series of novel 1-(substituted-phenyl)-3-(4,5-dihydro-1,3-oxazol-2-yl)-9H-β-carboline (8a-8i) and 1-(substituted-phenyl)-3-(5,6-dihydro-4H-1,3-oxazin-2-yl)-9H-β-carboline (9a-9h) derivatives, as well as their respective N-(chloroalkyl)-1-(substituted-phenyl)-9H-β-carboline-3-carboxamide precursors (6a-6i and 7a-7h), were synthesized and evaluated for their in vitro antileishmanial activity against promastigote and intracellular amastigote forms of Leishmania amazonensis. Compounds 8d, 8i, 9e and 9h exhibited significant activity for both promastigote and amastigote forms, with IC₅₀ (50% inhibitory concentration) values ranging from 2.9 to 23.0 µM. In addition, spin label electron paramagnetic resonance (EPR) spectroscopy studies were carried out for the most active compounds against L. amazonensis promastigotes. The studies indicated that the tested compounds cause strong stiffness in the parasite plasma membrane and are capable of inducing internal metalloproteins oxidation of the parasite, resulting in their cross-linking to skeletal proteins. Compounds 8d and 8i produced the largest effect, showing that the presence of oxazoline group at C-3 of β-carboline nucleus is important for antileishmanial activity.

Keywords:
β-carboline; 4,5-dihydro-1,3-oxazole; 5,6-dihydro-4H-1,3-oxazine; Leishmania amazonensis; electron paramagnetic resonance

Introduction

Leishmaniases are a group of diseases caused by protozoan parasites from more than 20 Leishmania species that cause a variety of clinical manifestations in humans, among them, there are three main forms: visceral (VL), cutaneous (CL) and mucocutaneous.¹ The Leishmania amazonensis species, for example, is responsible for the anergic diffuse cutaneous form and the cutaneous forms with disseminated lesions.² According to World Health Organization (WHO),¹ 97 countries and territories are endemic for leishmaniasis and it is estimated that between 600,000 to 1 million cases of CL occur worldwide annually. The current treatment for the leishmaniasis is mainly performed with pentavalent antimonials, amphotericin B, miltefosine and paromomycin. However, there is an increased incidence of treatment failure due to the toxicity and resistance exhibited by these drugs.³ Besides this, no vaccines against Leishmania infections are available.⁴ Therefore, it is of great importance to develop more active and less toxic compounds than the drugs used currently.

In the recent years, several studies have reported the antileishmanial activity of heterocyclic compounds,³ including β-carbolines alkaloids.^5-20 Ashok et al.,¹³ for example, described the antileishmanial activity against Leishmania infantum and Leishmania donovani of a series of (1-phenyl-9H-pyrido [3,4-b]indol-3-yl) (4-phenylpiperazin-1-yl)methanone derivatives. Among the derivatives assayed, compound I (Figure 1) displayed potent inhibition for both Leishmania species, with 50% effective concentration (EC₅₀) values ranging from 1.9 to 6.9 µM.

Figure 1
Structures of β-carbolines (I and II), oxazoline (III) and dihydrooxazine (IV) derivatives with antileishmanial activity.

In this context, our research group has already demonstrated the antileishmanial activity of β-carbolines containing substituents at 1- and 3-positions of the β-carboline nucleus.^14-20 In the works developed by Tonin et al.¹⁶ and Pedroso et al.,¹⁷ it was demonstrated the activity of N-alkyl-(1-phenylsubstituted-β-carboline)-3-carboxamides against promastigote, axenic amastigote and intracellular amastigote forms of Leishmania amazonensis. The compound with the N-benzyl-carboxamide group at C-3 was active against promastigote and axenic amastigote forms with IC₅₀ (50% inhibitory concentration) values of 2.6 and 1.0 µM, respectively,¹⁷ and killed L. amazonensis promastigotes through different cell death pathways, including apoptosis and autophagy.¹⁸ Recently, the antileishmanial activity of β-carboline-1,3,5-triazine hybrids was reported by Baréa et al.¹⁹ Among the compounds tested, the hybrid II (Figure 1) showed potent activity against the promastigote (IC₅₀ = 6.2 ± 1.4 µM, selectivity index (SI) = 23.5) and amastigote (IC₅₀ = 1.2 ± 0.5 µM, SI = 121.4) forms of L. amazonensis and exhibited low toxicity. Studies of action mechanism in promastigotes showed that compound II caused alterations in cell division cycle and an increase of lipid-storage bodies, leading the cells to death through various factors. The accumulation of lipid bodies may be associated with apoptotic cell death.¹⁹

Additionally, oxazoline and 5,6-dihydro-4H-1,3-oxazine heterocycles play an important role in organic synthesis, being present in the structure of various biologically active compounds.^20-27 For instance, the nitroimidazo-oxazole III (Figure 1) showed IC₅₀ of 0.03 µM against the amastigote form of L. donovani DD8 transfected with luciferase, and was identified, from a series of 72 nitroimidazoles evaluated, as a candidate for the oral treatment of visceral leishmaniasis. This compound showed also in vivo activity in both rat and hamster models.²⁵ The dihydrooxazine phenylpyridine IV (Figure 1) was effective for L. donovani mouse model and L. infantum hamster model, displaying optimal efficacy, pharmacokinetic and safety, leading to its selection as a new candidate for treatment of VL.²⁶

Considering the promising antileishmanial properties of β-carboline nucleus, the synthetic and biological importance of oxazoline and 5,6-dihydro-4H-1,3-oxazine rings, and the need to develop antileishmanial agents more effective, in this work we designed new 1-(substituted-phenyl)-β-carboline derivatives bearing oxazoline and 5,6-dihydro-4H-1,3-oxazine moieties at C-3 (Figure 2). The novel 1,3-disubstituted-β-carboline derivatives were evaluated against promastigote and intracellular amastigote forms of L. amazonensis and their cytotoxicity were determined. The antileishmanial activity of N-(chloroalkyl)-β-carboline intermediates, precursors of the proposed derivatives, was also evaluated in order to verify the importance of the heterocyclic ring at the 3-position of β-carboline nucleus.

Figure 2
Structures of novel β-carboline-oxazoline and β-carboline-(5,6-dihydro-4H-1,3-oxazine) derivatives proposed.

In addition, electron paramagnetic resonance (EPR) spectroscopy, associated with spin labeling method studies, was carried out for the most active compounds against L. amazonensis promastigotes. This analysis has been shown to be an important tool for analyzing the interaction of drugs or prototypes of drugs with parasite membranes. The literature describes the employment of this technique to evaluate the effects of miltefosine,^28,29 nerolidol^30,31 and parthenolide³² on L. amazonensis membrane, and of elatol on Trypanosoma cruzi.³³

Results and Discussion

Chemistry

Novel 1-(substituted-phenyl)-β-carboline derivatives bearing oxazoline (8a-8i) and 5,6-dihydro-4H-1,3-oxazine (9a-9h) moieties at C-3 were synthesized from the N-(chloroalkyl)-β-carboline-3-carboxamides 6a-6i and 7a-7h as shown in Scheme 1. The β-carboline nucleus was prepared from commercial L-tryptophan (1) according to the methodology described by our research group.^14-19 The L-tryptophan methyl ester (2) was subjected to the condensation reaction of Pictet-Spengler with different aldehydes in acid medium, providing the methyl 1-(substituted-phenyl)-1,2,3,4-tetrahydro-9H-β-carboline-3-carboxylates 3a-3i, which were oxidized with sulfur under reflux in xylene to the methyl 1-(substituted-phenyl)-9H-β-carboline-3-carboxylates 4a-4i. The basic hydrolysis reaction of 4a-4i provided the 1-(substituted-phenyl)-9H-β-carboline-3-carboxylic acids 5a-5i. The intermediates 5a-5i were subjected to the nucleophilic substitution reaction with 2-chloroethylamine or 3-chloropropylamine, using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as carboxylic acid activator, providing the N-(chloroalkyl)-β-carbolines 6a-6i and 7a-7i with yields in the range of 52 to 95%.

Scheme 1
Synthesis of derivatives 8a-8i and 9a-9h. Reagents and conditions: (a) MeOH, H2SO4 (cat), reflux, 48 h; (b) R1Ph-CHO, trifluoroacetic acid (TFA), CH2Cl2, room temperature (rt), 18-26 h; (c) sulfur, xylene, reflux, 48 h; (d) NaOH, MeOH/H2O (2:1), reflux, 6-18 h; (e) 2-chloroethylamine hydrochloride (for 6a-6i) or 3-chloropropylamine hydrochloride (for 7a-7i), Et3N, DMTMM, THF:MeOH (8:2); (f) (i) K2CO3, DMF, reflux, 12-20 h for 8a-8i; (ii) K2CO3, DMF, MW (100% power level), 5-9 min for 9a-9h.

The β-carboline-oxazoline 8a-8i derivatives were obtained in 46-93% yield (Figure 3) from the nucleophilic cyclo-O-alkylation of 6a-6i in refluxing dimethylformamide (DMF), using potassium carbonate as base. The intramolecular O-cyclization of 7a-7h using DMF under microwave irradiation and K₂CO₃, afforded the β-carboline-dihydrooxazine 9a-9h derivatives with yields in the range of 45 to 86% (Figure 3). The intermediate 7i was also submitted to cyclization reaction, under the similar conditions employed for 7a-7h; however, in this case, the product formed underwent decomposition during workup.

Figure 3
Yields and reaction times for preparation of 8a-8i and 9a-9h from cyclo-O-alkylation of 6a-6i and 7a-7h.

The β-carboline-oxazoline 8a-8i and β-carboline-dihydrooxazine 9a-9h derivatives were characterized by their spectral data (high-resolution mass spectrometry (HRMS), ¹H and ¹³C nuclear magnetic resonance (NMR)). The formation of the 8a-8i was supported by the absence of the signal at δ_H 8.54-9.01 referent to the NH of the carboxamide group and the presence of two triplets at δ_H 4.06-4.19 (CH₂, C-4”) and 4.50-4.59 (CH₂, C-5”), in ¹H NMR spectra. The presence of oxazoline ring was also confirmed by the signals at δ_C 54.4-55.1 (CH₂, C-4”) and 67.4-68.4 (CH₂, C-5”), in ¹³C NMR spectra. The 5,6-dihydro-4H-1,3-oxazine heterocycle at 3-positon of β-carboline nucleus in compounds 9a-9h were confirmed by the signals at δ_H 1.93-2.07 (quintet, CH₂), 3.55-3.74 (triplet, CH₂) and 3.83-4.51 (triplet, CH₂), in ¹H NMR spectra, and signals at δ_C 21.6-22.0 (CH₂), 42.1-43.0 (CH₂) and 64.7-65.7 (CH₂), in ¹³C NMR spectra.

Antileishmanial activity

The β-carboline-oxazolines 8a-8i and β-carboline-dihydrooxazines 9a-9h were evaluated in vitro against the promastigote form of L. amazonensis (Table 1). The compounds that showed IC₅₀ values greater than 100 µM were considered inactive. For the most active compounds against promastigotes, the antileishmanial activity for the intracellular amastigote form of L. amazonensis was also evaluated. The toxic effects on the host cells were determined by the selectivity index (SI). The SI for each active compound was calculated as the ratio between the cytotoxicity (CC₅₀) for macrophage J774-A1 cell lines and IC₅₀ against the promastigote and intracellular amastigote forms of L. amazonensis.

Table 1
Antileishmanial activity data for derivatives 8a-8i and 9a-9h against L. amazonensis

Compound	IC50pro / µM	IC50ama / µM	CC50 / µM	SIpro	SIama
8a	> 100	n.t	270.0 ± 28.3	n.d	n.d
8b	> 100	n.t	112.5 ± 24.7	n.d	n.d
8c	> 100	n.t	95.0 ± 5.0	n.d	n.d
8d	14.7 ± 6.7	10.5 ± 3.2	96.5 ± 10.6	6.6	9.2
8e	> 100	n.t	43.0 ± 18.4	n.d	n.d
8f	> 100	n.t	170.5 ± 28.9	n.d	n.d
8g	> 100	n.t	88.0 ± 10.6	n.d	n.d
8h	88.5 ± 10.6	n.t	249.5 ± 13.5	2.9	n.d
8i	23.0 ± 7.1	12.3 ± 3.1	30.5 ± 5.3	1.3	2.5
9a	27.5 ± 6.4	75.5 ± 6.7	67.5 ± 17.7	2.5	0.9
9b	53.5 ± 17.7	n.t	73.2 ± 16.3	1.4	n.d
9c	57.3 ± 11.2	n.t	120.0 ± 35.0	2.1	n.d
9d	58.0 ± 2.8	n.t	62.8 ± 24.7	1.1	n.d
9e	22.7 ± 2.1	2.9 ± 0.8	86.0 ± 12.1	3.8	29.7
9f	> 100	n.t	95.3 ± 30.7	n.d	n.d
9g	> 100	n.t	58.0 ±13.8	n.d	n.d
9h	21.3 ± 4.2	17.0 ± 1.4	39.5 ± 3.55	1.9	2.3
Miltefosinea	18.5 ± 1.1	2.4 ± 0.1	40.5 ± 1.7	2.2	16.9

• ^a
  Positive control.³⁴ IC_50pro: 50% inhibitory concentration against promastigotes; IC_50ama: 50% inhibitory concentration against intracellular amastigotes; CC₅₀: 50% cytotoxic concentration; SI_pro: selectivity index for promastigotes; SI_ama: selectivity index for intracellular amastigotes; n.t: not tested; n.d: not determined.

Analysis of the IC₅₀ values (Table 1) for β-carboline-oxazolines 8a-8i shows that the presence of chlorine and dimethylamino substituents, at 2- and 4-positions of phenyl group linked to C-1, led to the active compounds 8d and 8i, respectively. Compound 8d showed also better selectivity indices (SI) for both forms of L. amazonensis than for the host cells (Table 1), being the most promising compound in this series.

Concerning to 9a-9h series, most of β-carboline-dihydrooxazine derivatives showed moderate activity for L. amazonensis promastigotes, with IC₅₀ values ranging from 21.3 to 58.0 µM (Table 1). The derivatives 9a, 9e and 9h containing the phenyl, 4-chlorophenyl and 4-methoxyphenyl substituents, respectively, at C-1 of β-carboline nucleus, were the most active compounds for promastigote form, exhibiting IC₅₀ values in the range of 21.3 to 27.5 µM, similar to that of reference drug miltefosine.³⁴ These compounds were then evaluated against intracellular amastigote form of L. amazonensis and showed IC₅₀ values in the range of 2.9 to 75.5 µM (Table 1). The derivative 9e was the most promising, being 26 and 6 times more active than 9a and 9h, respectively. Besides that, 9e was 29.7 times more toxic for intracellular amastigotes than for macrophage J774-A1 cell lines, being a promising antileishmanial agent.

Comparison of the IC₅₀ data of the novel 1,3-disubstituted-β-carboline derivatives, 8a-8h and 9a-9h showed that except for compound 8d, the replacement of oxazoline by 5,6-dihydro-4H-1,3-oxazine ring led to an enhancement of the antileishmanial activity.

In this work, we also compared the antileishmanial activity of the β-carboline derivatives 8a-8i and 9a-9h with their intermediates N-(chloroalkyl)-β-carboline 6a-6i and 7a-7h, respectively (Table 2). Among the N-(chloroalkyl)-β-carbolines evaluated, only 6d was active, showing significant activity against both forms of L. amazonensis (IC_50pro = 2.2 ± 1.3 µM; IC_50ama = 6.3 ± 0.8 µM). These results demonstrate that the presence of oxazoline and dihydrooxazine rings in the 3-position of the β-carboline nucleus is important for antileishmanial activity.

Table 2
Antileishmanial activity data for compounds 6a-6i and 7a-7i against L. amazonensis

Compound	IC50pro / µM	IC50ama / µM	CC50 / µM	SIpro	SIama
6a	> 100	n.t	175.0 ± 35.4	n.d	n.d
6b	> 100	n.t	49.6 ± 16.3	n.d	n.d
6c	> 100	n.t	161.7 ± 7.6	n.d	n.d
6d	2.2± 1.3	6.3± 0.8	19.0± 6.2	8.7	3.0
6e	> 100	n.t	127.0 ± 32.5	n.d	n.d
6f	> 100	n.t	201.0 ± 9.9	n.d	n.d
6g	> 100	n.t	97.0 ± 4.2	n.d	n.d
6h	> 100	n.t	68.5 ± 10.6	n.d	n.d
6i	> 100	n.t	30.0 ± 3.0	n.d	n.d
7a	> 100	n.t	630.0 ± 169.0	n.d	n.d
7b	92.0 ± 4.05	n.t	83.3 ± 20.5	n.d	n.d
7c	> 100	n.t	70.7 ± 16.2	n.d	n.d
7d	> 100	n.t	< 10	n.d	n.d
7e	> 100	n.t	365.0 ± 120.2	n.d	n.d
7f	> 100	n.t	273.3 ± 59.6	n.d	n.d
7g	> 100	n.t	895.0 ± 148.0	n.d	n.d
7h	> 100	n.t	290.0 ± 88.2	n.d	n.d
7i	> 100	n.t	615.0 ± 49.5	n.d	n.d
Miltefosinea	18.5 ± 1.1	2.4 ± 0.1	40.5 ± 1.7	2.2	16.9

• ^a
  Positive control;³⁴ IC_50pro: 50% inhibitory concentration against promastigotes; IC_50ama: 50% inhibitory concentration against intracellular amastigotes; CC₅₀: 50% cytotoxic concentration; SI_pro: selectivity index for promastigotes; SI_ama: selectivity index for intracellular amastigotes; n.t: not tested; n.d: not determined.

Spin label EPR spectroscopy studies

In order to investigate the interaction of the most active compounds for the promastigote form of L. amazonensis with the parasite membrane, EPR spectroscopy associated with the spin labeling method studies were carried out for 6d, 8d, 8i, 9a, 9e and 9h. Figure 4 shows the EPR spectra of the spin label 5-doxyl-stearic acid (5-DSA) incorporated in Leishmania membranes for samples untreated and treated with the studied compounds. EPR spectra showed that all compounds cause increases in parameter 2A_// (outer hyperfine splitting) above the estimated experimental error (0.5 G), indicating decreases in molecular dynamics. In the treatment with 150 µM of compounds, some of them showed remarkable changes in the parasite membrane. Compounds 8d and 8i containing the oxazoline heterocycle at 3-position of β-carboline nucleus were the most effective for treatments at a concentration of 150 µM. However, we note that the compounds 6d, 9a, 9e and 9h can also cause high membrane stiffness at higher concentrations. In cell membrane the probe 5-DSA behaves as annular or boundary lipids that preferentially surround the hydrophobic surface of membrane proteins.³⁵ Because of these interactions with the transmembrane proteins, 5-DSA can monitor the dynamics at the periphery of proteins into the lipid bilayer. Thus, the changes in 5-DSA spectra caused by the compounds may be associated with changes in the membrane protein component.

Figure 4
EPR spectra of spin label 5-DSA incorporated in Leishmania amazonensis promastigotes membranes for samples of untreated cells (control) and treated with several compounds (150 µM). The values of the EPR parameter 2A_// (outer hyperfine splitting), which is given by the separation in magnetic field units between the first peak and the last inverted peak of the spectrum, are indicated. The estimated experimental error for the 2A_// parameter is 0.5 G. The total scan range of the magnetic field in each EPR spectrum was of 100 G (x axis) and the intensity (y axis) is in arbitrary units.

Spin label EPR spectroscopy indicated that the treatments of L. amazonensis promastigotes with the studied compounds cause strong stiffness in the parasite plasma membrane. These strong membrane changes, with changes in parameter 2A_// of ca. 5 G observed for two compounds at a relatively low concentration, cannot be explained by the simple presence of the molecules in the membrane, but must involve some oxidation process. Similar alterations in the EPR spectra of 5-DSA into plasma membrane were found in a previous study³⁶ for erythrocytes oxidized with hydrogen peroxide in phosphate buffer with azide (catalase inhibitor). EPR spectra of erythrocyte membrane spin-labeled with 5-DSA showed a change in parameter 2A_// of 57.5 G for untreated erythrocyte (control) to 60.5 G for erythrocyte treated with 200 µM H₂O₂. It has been shown that H₂O₂ induces the formation of cross-linking of hemoglobin to skeletal proteins in the membranes of human erythrocytes in an azide phosphate buffer, associated with a progressive alteration of the cell’s shape to echinocytic morphology, decreased cell deformability and increased phagocytosis.³⁷ Heme proteins were crucial for the occurrence of these cellular alterations, since they may be completely inhibited by previous exposure of red blood cells to carbon monoxide. Lipid peroxidation did not appear to be important because the antioxidant butylated hydroxytoluene decreased the fluorescent derivatives but did not prevent formation of the spectrin-Hb (hemoglobin) complex.³⁷ These observations suggest that the compounds tested are capable of inducing oxidation of internal metalloproteins of the parasite, resulting in their cross-linking to skeletal proteins.

Conclusions

Novel β-carboline-oxazoline 8a-8i and β-carboline-dihydrooxazine 9a-9h derivatives have been synthesized in moderate to good yields (45-93%) from the nucleophilic cyclo-O-alkylation of intermediates N-(chloroalkyl)-β-carboline-3-carboxamide 6a-6i and 7a-7h, respectively. Compounds 6d, 8d, 8i, 9e and 9h showed significant activity in L. amazonensis promastigotes (IC₅₀ values ranging from 2.2 to 23.0 µM), and also in intracellular amastigote forms (IC₅₀ values ranging from 2.9 to 17.0 µM). Compound 9e was the most active for intracellular amastigotes (IC₅₀ = 2.9 ± 0.8 µM) and showed also low toxicity, being 29.7 times more toxic for intracellular amastigotes than for macrophage J774-A1 cell lines.

The antileishmanial activity data showed that the presence of oxazoline and 5,6-dihydro-4H-1,3-oxazine moieties at C-3 of β-carboline nucleus led to an increase of compounds number with antileishmanial activity, in comparison to the N-(chloroalkyl)-β-carboline-3-carboxamide precursors. Comparison of the IC₅₀ data of the compounds 8a-8h and 9a-9h showed that except for compound 8d, the replacement of oxazoline by 5,6-dihydro-4H-1,3-oxazine ring led to an enhancement of the antileishmanial activity.

Spin label EPR spectroscopy studies indicated that the tested compounds cause strong stiffness in the parasite plasma membrane and are capable of inducing internal metalloproteins oxidation of the parasite, resulting in their cross-linking to skeletal proteins. Compounds 8d and 8i produced the largest effect, showing that the presence of oxazoline group at C-3 of β-carboline nucleus is important for antileishmanial activity. Further studies will be conducted with these compounds aiming a better understanding of their mechanisms of action. Compounds 8d and 8i are also strong candidates for in vivo studies in view to the development of new antileishmanial agents.

Experimental

General methods

All reagents were purchased from commercial suppliers, except the DMTMM that was synthesized according to the methodology described by Cronin et al.³⁸ and Kunishima et al.³⁹ The reactions were monitored by thin layer chromatography (TLC) conducted on Whatman TLC plates (silica gel 60 F₂₅₄). NMR spectra were recorded in a Varian spectrometer model Mercury plus BB at 300 (for ¹H) and 75 MHz (for ¹³C) and in a Bruker spectrometer model Avance III HD at 500 (for ¹H) and 125 MHz (for ¹³C), with deuterated solvents, chloroform (CDCl₃), methanol (CD₃OD) and dimethyl sulfoxide (DMSO-d₆), and tetramethylsilane (TMS) as internal standard. Mass spectra (electrospray ionization mass spectrometry (ESI-MS)) were recorded on Thermoelectron Corporation Focus-DSQ II spectrometer. Melting points were determined in Microquímica apparatus model MQAPF-301 and are uncorrected. Spin label 5-DSA was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Chemistry

Synthesis of 5a-5i

The intermediates 5a-5i were synthesized according to the protocol described previously by our research group.^16,17,19 To a suspension of 1-(substituted-phenyl)-β-carboline-3-carboxylates 4a-4i (1 mmol) in methanol-water (2:1), it was added 4 mmol of sodium hydroxide (0.16 g). The mixture was refluxed until complete consumption of 4a-4i (6-18 h). Then, the reaction mixture was cooled, treated with an HCl solution (2 M) until pH 5 and left on ice bath for 2 h. The precipitate formed was filtered and washed with distilled water. The β-carboline-carboxylic acids 5a-5i were obtained in yields in the range 59-97%.

Synthesis of N-(2-chloroethyl)-1-(substituted-phenyl)-9H-β-carboline-3-carboxamides (6a-6i) and N-(3-chloropropyl)-1-(substituted-phenyl)-9H-β-carboline-3-carboxamides (7a-7i)

To a solution of β-carboline-carboxylic acids 5a-5i (1 mmol) in tetrahydrofuran/methanol 8:2 (10 mL), it was added 1.5 mmol of 2-chloroethylamine hydrochloride (0.17 g) or 3-chloropropylamine hydrochloride (0.20 g), 1.5 mmol of triethylamine (0.21 mL) and 1.5 mmol of DMTMM (0.36 g). The reaction mixture was stirred at room temperature until complete consumption of 5a-5i (14 to 20 h). After this reaction time, the solvent was removed in a rotary evaporator and the residue solubilized in 1 mL of ethanol. Then, distilled water was added to the solution and the precipitate formed was filtered under vacuum and washed with distilled water. The compounds 6a-6i and 7a-7i were obtained in yields in the range of 52-95%.

N-(2-Chloroethyl)-1-phenyl-9H-β-carboline-3-carboxamide (6a)

Yield: 67%; mp 177-180 °C; ¹H NMR (300 MHz, CDCl₃) δ_H 3.76 (t, J 5.8 Hz, 2H, H-2”), 3.87 (q, J 5.8 Hz, 2H, H-1”), 7.35 (t, J 7.9 Hz, 1H, H-6), 7.50-7.64 (m, 5H, H-7, H-8, H-3’, H-4’, H-5’), 7.99 (d, J 7.2 Hz, 2H, H-2’, H-6’), 8.18 (d, J 7.9 Hz, 1H, H-5), 8.66 (t, 1H, J 5.8 Hz, CONH), 8.87 (s, H-4), 8.93 (s, 1H, NH, H-9); ¹³C NMR (75 MHz, CDCl₃) δ_C 41.3 (CH₂, C-1”), 43.9 (CH₂, C-2”), 111.8 (CH, C-8), 113.6 (CH, C-4), 121.1 (CH, C-6), 122.2 (CH, C-5), 122.2 (C₀, C-4b), 128.2 (2CH, C-2’, C-6’), 129.0 (CH, C-4’), 129.3 (CH, C-7), 129.3 (CH, C-3’, C-5’), 130.6 (C₀, C-4a), 134.9 (C₀, C-9a), 137.8 (C₀, C-1’), 140.0 (C₀, C-3), 140.7 (C₀, C-1), 141.1 (C₀, C-8a), 165.8 (C=O); HRMS-ESI m/z, calcd. for C₂₀H₁₇ClN₃O [M + H]⁺: 350.1055, found: 350.1049.

N-(2-Chloroethyl)-1-(2-fluorophenyl)-9H-β-carboline-3-carboxamide (6b)

Yield: 75%; mp 171-172 °C; ¹H NMR (300 MHz, CDCl₃) δ_H 3.77 (t, J 5.8 Hz, 2H, H-2”), 3.89 (q, J 5.8 Hz, 2H, H-1”), 7.29-7.44 (m, 3H, H-6, H-3’, H-6’), 7.50-7.63 (m, 3H, H-4’, H-7, H-8), 7.90 (td, J 7.6, 1.7 Hz, 1H, H-5’), 8.23 (d, J 7.9 Hz, 1H, H-5), 8.58-8.63 (m, 2H, CONH, NH, H-9), 8.95 (s, 1H, H-4); ¹³C NMR (75 MHz, CDCl₃) δ_C 41.2 (CH₂, C-1”), 43.9 (CH₂, C-2”), 111.8 (CH, C-8), 114.1 (CH, C-4), 116.3 (CH, d, J 22.5 Hz, C-3’), 121.0 (CH, C-6), 122.0 (C₀, C-4b), 122.2 (CH, C-5), 125.2 (CH, C-6’), 125.3 (C₀, C-4a), 129.1 (CH, C-7), 130.6 (C₀, C-1’), 131.2 (C₀, d, J 8.4 Hz, C-4’), 132.3 (CH, d, J 3.7 Hz, C-5’), 135.6 (C₀, C-9a), 136.3 (C₀, C-3), 140.1 (C₀, C-1), 140.6 (C₀, C-8a), 159.9 (C₀, d, J 246 Hz, 1C, C-2’), 165.6 (C=O); HRMS-ESI m/z, calcd. for C₂₀H₁₆ClFN₃O [M + H]⁺: 368.0960, found: 368.0963.

N-(2-Chloroethyl)-1-(4-fluorophenyl)-9H-β-carboline-3-carboxamide (6c)

Yield: 91%; mp 164-167 °C; ¹H NMR (300 MHz, CDCl₃/CD₃OD) δ_H 3.77 (t, J 6.6 Hz, 2H, H-2”), 3.87 (m, 2H, H-1”), 7.27-7.36 (m, 3H, H-6, H-3’, H-5’), 7.58 (m, 2H, H-7, H-8), 7.99 (dd, J 8.7, 5.3 Hz, 2H, H-2’, H-6’), 8.17 (d, J 7.8 Hz, 1H, H-5), 8.67 (t, J 5.8 Hz, CONH), 8.82 (s, 1H, H-4), 9.81 (s, 1H, NH, H-9); ¹³C NMR (75 MHz, CDCl₃/CD₃OD) δ_C 41.2 (CH₂, C-1”), 43.8 (CH₂, C-2”), 112.0 (CH, C-8), 113.6 (CH, C-4), 116.1 (d, J 21.9 Hz, 2CH, C-3’, C-5’), 120.9 (CH, C-6), 122.0 (CH, C-5), 122.1 (C₀, C-4b), 128.9 (CH, C-7), 130.2 (d, J 8.5 Hz, 2 CH, C-2’, C-6’), 130.7 (C₀, C-4a), 134.0 (C₀, C-9a), 135.0 (C₀, C-1’), 139.4 (C₀, C-3), 140.3 (C₀, C-1), 141.2 (C₀, C-8a), 163.3 (d, J 247.7 Hz, C₀, C-4’), 166.1 (C=O); HRMS-ESI m/z, calcd. for C₂₀H₁₆ClFN₃O [M + H]⁺: 368.0960, found: 368.0957.

N-(2-Chloroethyl)-1-(2-chlorophenyl)-9H-β-carboline-3-carboxamide (6d)

Yield: 94%; mp 246-248 °C; ¹H NMR (300 MHz, CDCl₃) δ_H 3.76 (t, J 5.8 Hz, 2H, H-2”), 3.86 (q, J 5.8 Hz, 2H, H-1”), 7.37 (t, J 7.8 Hz, 1H, H-6), 7.47-7.68 (m, 6H, H-7, H-8, H-3’, H-4’, H-5’, H-6’), 8.23 (d, J 7.8 Hz, 1H, H-5), 8.54 (m, 2H, CONH, NH, H-9), 8.95 (s, 1H, H-4); ¹³C NMR (75 MHz, CDCl₃) δ_C 41.4 (CH₂, C-1”), 43.7 (CH₂, C-2”), 111.8 (CH, C-8), 114.2 (CH, C-4), 121.0 (CH, C-6), 122.1 (C₀, C-4b), 122.2 (CH, C-5), 127.4 (CH, C-7), 129.1 (CH, C-5’), 130.2 (C₀, C-4a), 130.4 (CH, C-3’), 130.5 (CH, C-6’), 131.9 (CH, C-4’), 133.0 (C₀, C-2’), 135.4 (C₀, C-9a), 136.3 (C₀, C-1’), 139.5 (C₀, C-3), 139.8 (C₀, C-1), 140.7 (C₀, C-8a), 165.7 (C=O); HRMS-ESI m/z, calcd. for C₂₀H₁₆Cl₂N₃O [M + H]⁺: 384.0665, found: 384.0665.

N-(2-Chloroethyl)-1-(4-chlorophenyl)-9H-β-carboline-3-carboxamide (6e)

Yield: 68%; mp 269-272 °C; ¹H NMR (300 MHz, CDCl₃/CD₃OD) δ_H 3.78 (t, J 5.8 Hz, 2H, H-2”), 3.88 (t, J 5.8 Hz, 2H, H-1”), 7.33-7.36 (m, 1H, H-6), 7.59 (m, 4H, H-7, H-8, H-3’, H-5’), 7.98 (d, J 8.1 Hz, 2H, H-2’, H-6’), 8.20 (d, J 7.9 Hz, 1H, H-5), 8.83 (s, 1H, H-4); ¹³C NMR (75 MHz, CDCl₃/CD₃OD) δ_C 41.3 (CH₂, C-1”), 43.8 (CH₂, C-2”), 112.2 (CH, C-8), 113.8 (CH, C-4), 120.9 (CH, C-6), 122.0 (CH, C-5), 122.0 (C₀, C-4b), 129.0 (CH, C-7), 129.3 (2CH, C-3’, C-5’), 129.8 (2CH, C-2’, C-6’), 130.8 (C₀, C-4a), 135.0 (C₀, C-4’), 135.2 (C₀, C-9a), 136.4 (C₀, C-1’), 139.3 (C₀, C-3), 140.1 (C₀, C-1), 141.4 (C₀, C-8a), 166.3 (C=O); HRMS-ESI m/z, calcd. for C₂₀H₁₆Cl₂N₃O [M + H]⁺: 384.0665, found: 384.0673.

N-(2-Chloroethyl)-1-(3-nitrophenyl)-9H-β-carboline-3-carboxamide (6f)

Yield: 72%; mp 186-190 °C; ¹H NMR (300 MHz, DMSO-d₆) δ_H 3.70-3.84 (m, 4H, H-1”, H-2”), 7.35 (td, J 7.4, 1.1 Hz, 1H, H-6), 7.61-7.71 (m, 2H, H-7, H-8), 7.96 (t, J 8.0 Hz, 1H, H-5’), 8.40-8.48 (m, 2H, H-5, H-4’), 8.59 (dt, J 8.0, 1.1 Hz, 1H, H-6’), 8.86 (t, J 1.9 Hz, 1H, H-2’), 8.93 (s, 1H, H-4), 9.01 (t, J 5.8 Hz, 1H, CONH), 12.06 (s, 1H, NH, H-9); ¹³C NMR (75 MHz, DMSO-d₆) δ_C 41.0 (CH₂, C-1”), 43.3 (CH₂, C-2”), 112.6 (CH, C-8), 114.1 (CH, C-4), 120.5 (CH, C-6), 121.2 (C₀, C-4b), 122.3 (CH, C-5), 123.5 (CH, C-2’), 123.6 (CH, C-4’), 129.0 (CH, C-7), 130.4 (C₀, C-4a), 130.4 (CH, C-5’), 134.5 (CH, C-6’), 135.4 (C₀, C-9a), 138.3 (C₀, C-1’), 138.9 (C₀, C-3), 139.7 (C₀, C-1), 141.7 (C₀, C-8a), 148.3 (C₀, C-3’), 164.9 (C=O); HRMS-ESI m/z, calcd. for C₂₀H₁₆ClN₄O₃ [M + H]⁺: 395.0905, found: 395.0905.

N-(2-Chloroethyl)-1-(4-nitrophenyl)-9H-β-carboline-3-carboxamide (6g)

Yield: 79%; mp > 279 °C (decomp.); ¹H NMR (300 MHz, DMSO-d₆) δ_H 3.73-3.84 (m, 4H, H-1”, H-2”), 7.35 (td, J 7.4, 1.1 Hz, 1H, H-6), 7.61-7.72 (m, 2H, H-7, H-8), 8.45-8.47 (m, 5H, H-5, H-2’, H-3’, H-5’, H-6’), 8.94 (s, 1H, H-4), 9.01 (t, J 5.8 Hz, CONH), 12.07 (s, 1H, NH, H-9); ¹³C NMR (75 MHz, DMSO-d₆) δ_C 41.0 (CH₂, C-1”), 43.2 (CH₂, C-2”), 112.6 (CH, C-8), 114.3 (CH, C-4), 120.5 (CH, C-6), 121.1 (CH, C-5), 122.3 (C₀, C-4b), 123.8 (2CH, C-3’, C-5’), 129.1 (CH, C-7), 130.1 (2CH, C-2’, C-6’), 130.7 (C₀, C-4a), 134.6 (C₀, C-9a), 138.0 (C₀, C-1’), 139.7 (C₀, C-3), 141.7 (C₀, C-1), 143.6 (C₀, C-8a), 147.5 (C₀, C-4’), 164.8 (C=O); HRMS-ESI m/z, calcd. for C₂₀H₁₆ClN₄O₃ [M + H]⁺: 395.0905, found: 395.0905.

N-(2-Chloroethyl)-1-(4-methoxyphenyl)-9H-β-carboline-3-carboxamide (6h)

Yield: 82%; mp 169-173 °C; ¹H NMR (300 MHz, CDCl₃) δ_H 3.78 (t, J 5.8 Hz, 2H, H-2”), 3.86-3.91 (m, 2H, H-1”), 3.91 (s, 3H, OCH₃), 7.14 (d, J 8.7 Hz, 2H, H-3’, H-5’), 7.36 (t, J 7.9 Hz, 1H, H-6), 7.55-7.62 (m, 2H, H-7, H-8), 7.95 (d, J 8.7 Hz, 2H, H-2’, H-6’), 8.20 (d, J 7.9 Hz, 1H, H-5), 8.67 (t, J 5.9 Hz, CONH), 8.84 (s, 1H, H-4), 8.88 (s, 1H, NH, H-9); ¹³C NMR (75 MHz, CDCl₃) δ_C 41.3 (CH₂, C-1”), 43.9 (CH₂, C-2”), 55.5 (OCH₃), 111.8 (CH, C-8), 113.2 (CH, C-4), 114.7 (2CH, C-3’, C-5’), 121.0 (CH, C-6), 122.1 (CH, C-5), 122.3 (C₀, C-4b), 128.8 (CH, C-7), 129.5 (2CH, C-2’, C-6’), 130.3 (C₀, C-4a), 130.4 (C₀, C-9a), 134.8 (C₀, C-1’), 139.9 (C₀, C-3), 140.7 (C₀, C-1), 141.1 (C₀, C-8a), 160.5 (C₀, C-4’), 165.9 (C=O); HRMS-ESI m/z, calcd. for C₂₁H₁₉ClN₃O₂ [M + H]⁺: 380.1160, found: 380.1153.

N-(2-Chloroethyl)-1-[4-(dimethylamino)phenyl]-9H-β-carboline-3-carboxamide (6i)

Yield: 85%; mp > 304 °C (decomp.); ¹H NMR (300 MHz, CDCl₃) δ_H 3.04 (s, 6H, N(CH₃)₂), 3.76 (t, J 6.0 Hz, 2H, H-2”), 3.88 (q, J 6.0 Hz, 2H, H-1”), 6.90 (d, J 8.8 Hz, 2H, H-3’, H-5’), 7.33 (ddd, J 7.9, 5.3, 2.7 Hz, 1H, H-6), 7.53-7.57 (m, 2H, H-7, H-8), 7.89 (d, J 8.8 Hz, 2H, H-2’, H-6’), 8.18 (d, J 7.9 Hz, 1H, H-5), 8.72 (t, J 6.0 Hz, CONH), 8.78 (s, 1H, H-4), 8.90 (s, 1H, NH, H-9); ¹³C NMR (75 MHz, CDCl₃) δ_C 40.3 (N(CH₃)₂), 41.3 (CH₂, C-1”), 43.9 (CH₂, C-2”), 111.7 (CH, C-8), 112.5 (2CH, C-3’, C-5’), 112.5 (CH, C-4), 120.8 (CH, C-6), 122.1 (CH, C-5), 122.4 (C₀, C-4b), 125.4 (C₀, C-4a), 128.6 (CH, C-7), 129.0 (2CH, C-2’, C-6’), 130.0 (C₀, C-9a), 134.7 (C₀, C-1’), 139.8 (C₀, C-3), 140.6 (C₀, C-1), 141.8 (C₀, C-8a), 151.0 (C₀, C-4’), 166.1 (C=O); HRMS-ESI m/z, calcd. for C₂₂H₂₂ClN₄O [M + H]⁺: 393.1477, found: 393.1465.

N-(3-Chloropropyl)-1-phenyl-9H-β-carboline-3-carboxamide (7a)

Yield: 82%; mp 161-164 °C; ¹H NMR (500 MHz, CDCl₃) δ_H 2.16 (qt, J 6.4 Hz, 2H, H-2”), 3.65-3.70 (m, 4H, H-1”, H-3”), 7.34 (t, J 7.8 Hz, 1H, H-6), 7.51-7.62 (m, 5H, H-7, H-8, H-3’, H-4’, H-5’), 7.97 (d, J 7.3 Hz, 2H, H-2’, H-6’), 8.18 (d, J 7.8 Hz, 1H, H-5), 8.42 (s, 1H, CONH), 8.87 (s, H-4), 8.97 (s, 1H, NH, H-9); ¹³C NMR (125 MHz, CDCl₃) δ_C 32.6 (CH₂, C-2”), 37.0 (CH₂, C-1”), 42.7 (CH₂, C-3”), 111.8 (CH, C-8), 113.5 (CH, C-4), 121.0 (CH, C-6), 122.2 (CH, C-5), 122.3 (C₀, C-4b), 128.2 (2CH, C-2’, C-6’), 128.9 (CH, C-4’), 129.3 (CH, C-7), 129.3 (CH, C-3’, C-5’), 130.6 (C₀, C-4a), 134.8 (C₀, C-9a), 137.9 (C₀, C-1’), 140.3 (C₀, C-3), 140.7 (C₀, C-1), 141.1 (C₀, C-8a), 165.9 (C=O); HRMS-ESI m/z, calcd. for C₂₁H₁₉ClN₃O [M + H]⁺: 364.1211, found: 364.1181.

N-(3-Chloropropyl)-1-(2-fluorophenyl)-9H-β-carboline-3-carboxamide (7b)

Yield: 52%; mp 173-174 °C; ¹H NMR (300 MHz, CDCl₃) δ_H 2.17 (qt, J 6.6 Hz, 2H, H-2”), 3.65-3.73 (m, 4H, H-1”, H-3”), 7.28-7.43 (m, 3H, H-6, H-3’, H-6’), 7.50-7.62 (m, 3H, H-4’, H-7, H-8), 7.88 (td, J 7.6, 1.8 Hz, 1H, H-5’), 8.22 (d, J 7.9 Hz, 1H, H-5), 8.36 (t, J 6.1 Hz, 1H, CONH), 8.64 (s, 1H, NH, H-9), 8.94 (s, 1H, H-4); ¹³C NMR (75 MHz, CDCl₃) δ_C 32.6 (CH₂, C-2”), 37.0 (CH₂, C-1”), 42.7 (CH₂, C-3”), 111.8 (CH, C-8), 114.0 (CH, C-4), 116.3 (CH, d, J 21.5 Hz, C-3’), 121.0 (CH, C-6), 122.0 (C₀, C-4b), 122.2 (CH, C-5), 125.2 (CH, C-6’), 125.3 (C₀, C-4a), 129.1 (CH, C-7), 130.6 (C₀, C-1’), 131.1 (CH, d, J 7.5 Hz, C-4’), 132.3 (CH, d, J 3.8 Hz, C-5’), 135.5 (C₀, C-9a), 136.2 (C₀, C-3), 140.4 (C₀, C-1), 140.6 (C₀, C-8a), 159.9 (C₀, d, J 246 Hz, 1C, C-2’), 165.7 (C=O); HRMS-ESI m/z, calcd. for C₂₁H₁₈ClFN₃O [M + H]⁺: 382.1117, found: 382.1085.

N-(3-Chloropropyl)-1-(4-fluorophenyl)-9H-β-carboline-3-carboxamide (7c)

Yield: 92%; mp 131-133 °C; ¹H NMR (300 MHz, CDCl₃) δ_H 2.17 (qt, J 6.5 Hz, 2H, H-2”), 3.65-3.73 (m, 4H, H-1”, H-3”), 7.28-7.39 (m, 3H, H-6, H-3’, H-5’), 7.55-7.62 (m, 2H, H-7, H-8), 7.97 (dd, J 8.5, 5.3 Hz, 2H, H-2’, H-6’), 8.19 (d, J 8.0 Hz, 1H, H-5), 8.38 (t, J 6.0 Hz, CONH), 8.83 (s, 1H, NH, H-9), 8.87 (s, 1H, H-4); ¹³C NMR (75 MHz, CDCl₃) δ_C 32.6 (CH₂, C-2”), 37.0 (CH₂, C-1”), 42.7 (CH₂, C-3”), 111.8 (CH, C-8), 113.6 (CH, C-4), 116.4 (d, J 21.3 Hz, 2CH, C-3’, C-5’), 121.2 (CH, C-6), 122.2 (CH, C-5), 122.3 (C₀, C-4b), 129.1 (CH, C-7), 130.1 (d, J 8.5 Hz, 2CH, C-2’, C-6’), 130.8 (C₀, C-4a), 134.0 (C₀, C-1’), 134.7 (C₀, C-9a), 140.1 (C₀, C-3), 140.4 (C₀, C-1), 140.7 (C₀, C-8a), 163.3 (d, J 247.7 Hz, C₀, C-4’), 165.8 (C=O); HRMS-ESI m/z, calcd. for C₂₁H₁₈ClFN₃O [M + H]⁺: 382.1117, found: 382.1091.

N-(3-Chloropropyl)-1-(2-chlorophenyl)-9H-β-carboline-3-carboxamide (7d)

Yield: 95%; mp 195-198 °C; ¹H NMR (300 MHz, CDCl₃) δ_H 2.14 (qt, J 6.5 Hz, 2H, H-2”), 3.62-3.68 (m, 4H, H-1”, H-3”), 7.35 (t, J 7.8 Hz, 1H, H-6), 7.46-7.65 (m, 6H, H-7, H-8, H-3’, H-4’, H-5’, H-6’), 8.21 (d, J 7.8 Hz, 1H, H-5), 8.31 (t, J 5.5 Hz, 1H, CONH), 8.63 (s, 1H, NH, H-9), 8.92 (s, 1H, H-4); ¹³C NMR (75 MHz, CDCl₃) δ_C 32.6 (CH₂, C-2”), 36.9 (CH₂, C-1”), 42.6 (CH₂, C-3”), 111.8 (CH, C-8), 114.1 (CH, C-4), 121.0 (CH, C-6), 122.1 (C₀, C-4b), 122.2 (CH, C-5), 127.4 (CH, C-7), 129.1 (CH, C-5’), 130.2 (C₀, C-4a), 130.4 (CH, C-3’), 130.5 (CH, C-6’), 131.9 (CH, C-4’), 133.0 (C₀, C-2’), 135.4 (C₀, C-9a), 136.3 (C₀, C-1’), 139.4 (C₀, C-3), 140.0 (C₀, C-1), 140.8 (C₀, C-8a), 165.8 (C=O); HRMS-ESI m/z, calcd. for C₂₁H₁₈Cl₂N₃O [M + H]⁺: 398.0821, found: 398.0782.

N-(3-Chloropropyl)-1-(4-chlorophenyl)-9H-β-carboline-3-carboxamide (7e)

Yield: 93%; mp 175-178 °C; ¹H NMR (500 MHz, CDCl₃) δ_H 2.17 (qt, J 6.4 Hz, 2H, H-2”), 3.66-3.72 (m, 4H, H-1”, H-3”), 7.36 (t, J 7.8 Hz, 1H, H-6), 7.56-7.58 (m, 4H, H-7, H-8, H-3’, H-5’), 7.92 (d, J 7.8 Hz, 2H, H-2’, H-6’), 8.17 (d, J 7.8 Hz, 1H, H-5), 8.37 (s, 1H, CONH), 8.86 (s, 1H, H-4), 8.91 (s, 1H, NH, H-9); ¹³C NMR (125 MHz, CDCl₃) δ_C 32.5 (CH₂, C-2”), 37.0 (CH₂, C-1”), 42.7 (CH₂, C-3”), 111.9 (CH, C-8), 113.7 (CH, C-4), 121.2 (CH, C-6), 122.2 (CH, C-5), 122.2 (C₀, C-4b), 129.1 (CH, C-7), 129.5 (4CH, C-3’, C-5’, C-2’, C-6’), 130.9 (C₀, C-4a), 134.7 (C₀, C-4’), 135.3 (C₀, C-9a), 136.3 (C₀, C-1’), 139.8 (C₀, C-3), 140.4 (C₀, C-1), 140.8 (C₀, C-8a), 165.7 (C=O); HRMS-ESI m/z, calcd. for C₂₁H₁₈Cl₂N₃O [M + H]⁺: 398.0821, found: 398.0791.

N-(3-Chloropropyl)-1-(3-nitrophenyl)-9H-β-carboline-3-carboxamide (7f)

Yield: 93%; mp 203-206 °C; ¹H NMR (300 MHz, DMSO-d₆) δ_H 2.07 (qt, J 6.5 Hz, 2H, H-2”), 3.53 (q, J 6.5 Hz, 2H, H-1”), 3.73 (t, J 6.5 Hz, 2H, H-3”), 7.34 (t, J 7.2 Hz, 1H, H-6), 7.60-7.70 (m, 2H, H-7, H-8), 7.94 (t, J 7.9 Hz, 1H, H-5’), 8.40-8.46 (m, 2H, H-5, H-4’), 8.60 (d, J 7.9 Hz, 1H, H-6’), 8.86-8.92 (m, 3H, H-2’, H-4, CONH), 12.03 (s, 1H, NH, H-9); ¹³C NMR (75 MHz, DMSO-d₆) δ_C 32.5 (CH₂, C-2”), 36.7 (CH₂, C-1”), 43.4 (CH₂, C-3”), 112.5 (CH, C-8), 113.9 (CH, C-4), 120.4 (CH, C-6), 121.2 (C₀, C-4b), 122.2 (CH, C-5), 123.5 (CH, C-2’), 123.5 (CH, C-4’), 128.9 (CH, C-7), 130.3 (CH, C-5’), 130.4 (C₀, C-4a), 134.4 (C₀, C-9a), 135.4 (CH, C-6’), 138.2 (C₀, C-1’), 138.9 (C₀, C-3), 140.1 (C₀, C-1), 141.6 (C₀, C-8a), 148.3 (C₀, C-3’), 164.8 (C=O); HRMS-ESI m/z, calcd. for C₂₁H₁₈ClN₄O₃ [M + H]⁺: 409.1062, found: 409.1032.

N-(3-Chloropropyl)-1-(4-nitrophenyl)-9H-β-carboline-3-carboxamide (7g)

Yield: 83%; mp 233-236 °C; ¹H NMR (300 MHz, DMSO-d₆) δ_H 2.08 (qt, J 6.6 Hz, 2H, H-2”), 3.53 (q, J 6.6 Hz, 2H, H-1”), 3.73 (t, J 6.6 Hz, 2H, H-3”), 7.34 (t, J 7.1 Hz, 1H, H-6), 7.60-7.71 (m, 2H, H-7, H-8), 8.43-8.50 (m, 5H, H-5, H-2’, H-3’, H-5’, H-6’), 8.88-8.91 (m, 2H, H-4, CONH), 12.03 (s, 1H, NH, H-9); ¹³C NMR (75 MHz, DMSO-d₆) δ_C 32.6 (CH₂, C-2”), 36.7 (CH₂, C-1”), 43.3 (CH₂, C-3”), 112.6 (CH, C-8), 114.1 (CH, C-4), 120.5 (CH, C-6), 121.1 (C₀, C-4b), 122.2 (CH, C-5), 123.7 (2CH, C-3’, C-5’), 129.0 (CH, C-7), 130.1 (2CH, C-2’, C-6’), 130.7 (C₀, C-4a), 134.5 (C₀, C-9a), 137.9 (C₀, C-1’), 140.1 (C₀, C-3), 141.7 (C₀, C-1), 143.7 (C₀, C-8a), 147.4 (C₀, C-4’), 164.8 (C=O); HRMS-ESI m/z, calcd. for C₂₁H₁₈ClN₄O₃ [M + H]⁺: 409.1062, found: 409.1040.

N-(3-Chloropropyl)-1-(4-methoxyphenyl)-9H-β-carboline-3-carboxamide (7h)

Yield: 85%; mp 192-194 °C; ¹H NMR (500 MHz, CDCl₃) δ_H 2.17 (qt, J 6.5 Hz, 2H, H-2”), 3.66-3.71 (m, 4H, H-1”, H-3”), 3.90 (s, 3H, OCH₃), 7.13 (d, J 8.4 Hz, 2H, H-3’, H-5’), 7.35 (t, J 7.9 Hz, 1H, H-6), 7.55-7.59 (m, 2H, H-7, H-8), 7.92 (d, J 8.4 Hz, 2H, H-2’, H-6’), 8.19 (d, J 7.9 Hz, 1H, H-5), 8.42 (t, J 6.0 Hz, CONH), 8.84 (s, 1H, H-4), 8.87 (s, 1H, NH, H-9); ¹³C NMR (125 MHz, CDCl₃) δ_C 32.6 (CH₂, C-2”), 37.0 (CH₂, C-1”), 42.7 (CH₂, C-3”), 55.5 (OCH₃), 111.8 (CH, C-8), 113.1 (CH, C-4), 114.7 (2CH, C-3’, C-5’), 121.0 (CH, C-6), 122.2 (CH, C-5), 122.4 (C₀, C-4b), 128.8 (CH, C-7), 129.5 (2CH, C-2’, C-6’), 130.3 (C₀, C-4a), 130.4 (C₀, C-9a), 134.7 (C₀, C-1’), 140.3 (C₀, C-3), 140.7 (C₀, C-1), 141.0 (C₀, C-8a), 160.5 (C₀, C-4’), 165.9 (C=O); HRMS-ESI m/z, calcd. for C₂₂H₂₁ClN₃O₂ [M + H]⁺: 394.1317, found: 394.1338.

N-(3-Chloropropyl)-1-[4-(dimethylamino)phenyl]-9H-β-carboline-3-carboxamide (7i)

Yield: 62%; mp 198-202 °C; ¹H NMR (300 MHz, CDCl₃) δ_H 2.14 (qt, J 6.6 Hz, 2H, H-2”), 2.99 (s, 6H, N(CH₃)₂), 3.63-3.70 (m, 4H, H-1”, H-3”), 6.85 (d, J 8.8 Hz, 2H, H-3’, H-5’), 7.31 (td, J 7.9, 1.3 Hz, 1H, H-6), 7.51-7.59 (m, 2H, H-7, H-8), 7.88 (d, J 8.8 Hz, 2H, H-2’, H-6’), 8.14 (d, J 7.9 Hz, 1H, H-5), 8.48 (t, J 6.2 Hz, CONH), 8.76 (s, 1H, H-4), 9.16 (s, 1H, NH, H-9); ¹³C NMR (75 MHz, CDCl₃) δ_C 32.6 (CH₂, C-2”), 36.9 (CH₂, C-1”), 40.2 (N(CH₃)₂), 42.7 (CH₂, C-3”), 111.8 (CH, C-8), 112.3 (2CH, C-3’, C-5’), 112.4 (CH, C-4), 120.6 (CH, C-6), 122.0 (CH, C-5), 122.3 (C₀, C-4b), 125.3 (C₀, C-4a), 128.4 (CH, C-7), 129.0 (2CH, C-2’, C-6’), 130.0 (C₀, C-9a), 134.6 (C₀, C-1’), 139.9 (C₀, C-3), 140.7 (C₀, C-1), 141.7 (C₀, C-8a), 150.9 (C₀, C-4’), 166.1 (C=O); HRMS-ESI m/z, calcd. for C₂₃H₂₄ClN₄O [M + H]⁺: 407.1633, found: 407.1650.

Synthesis of 1-(substituted-phenyl)-3-(4,5-dihydro-1,3-oxazol-2-yl)-9H-β-carboline (8a-8i)

To a solution of intermediates 2-chloroethyl-β-carboline-3-carboxamides 6a-6i (0.3 mmol) in DMF (3 mL), it was added 2 equivalents of potassium carbonate (0.08 g). The reaction mixture was refluxed until complete consumption of intermediates (14-20 h). After this time, the solution was cooled, treated with 5 mL of distilled water and left on ice bath for 3 h. The solid formed was filtered under vacuum, washed with distilled water, and recrystallized with methanol. The derivatives 8a-8i were obtained in yields in the range of 46-93%.

1-Phenyl-3-(4,5-dihydro-1,3-oxazol-2-yl)-9H-β-carboline (8a)

Yield: 85%; mp 224-226 °C; ¹H NMR (300 MHz, CDCl₃) δ_H 4.18 (t, J 9.3 Hz, 2H, H-4”), 4.59 (t, J 9.3 Hz, 2H, H-5”), 7.35 (t, J 7.9 Hz, 1H, H-6), 7.44-7.61 (m, 5H, H-7, H-8, H-3’, H-4’, H-5’), 7.96 (d, J 7.2 Hz, 2H, H-2’, H-6’), 8.18 (d, J 7.9 Hz, 1H, H-5), 8.76 (s, H-4), 8.82 (s, 1H, NH, H-9); ¹³C NMR (75 MHz, CDCl₃) δ_C 55.0 (CH₂, C-4”), 68.2 (CH₂, C-5”), 111.8 (CH, C-8), 115.1 (CH, C-4), 120.9 (CH, C-6), 122.0 (CH, C-5), 122.1 (C₀, C-4b), 128.4 (2CH, C-2’, C-6’), 128.8 (CH, C-4’), 129.0 (CH, C-7), 129.1 (CH, C-3’, C-5’), 129.9 (C₀, C-4a), 134.4 (C₀, C-9a), 136.9 (C₀, C-3), 138.0 (C₀, C-1’), 140.6 (C₀, C-8a), 142.9 (C₀, C-1), 165.0 (C₀, C-2”); HRMS-ESI m/z, calcd. for C₂₀H₁₆N₃O [M + H]⁺: 314.1288, found: 314.1245.

1-(2-Fluorophenyl)-3-(4,5-dihydro-1,3-oxazol-2-yl)-9H-β-carboline (8b)

Yield: 46%; mp 261-263 °C; ¹H NMR (300 MHz, CDCl₃) δ_H 4.17 (t, J 9.5 Hz, 2H, H-4”), 4.58 (t, J 9.5 Hz, 2H, H-5”), 7.20-7.36 (m, 3H, H-6, H-3’, H-6’), 7.42-7.60 (m, 3H, H-4’, H-7, H-8), 7.87 (td, J 7.5, 1.4 Hz, 1H, H-5’), 8.17 (d, J 7.8 Hz, 1H, H-5), 8.69 (s, 1H, NH, H-9), 8.80 (s, 1H, H-4); ¹³C NMR (75 MHz, CDCl₃) δ_C 55.0 (CH₂, C-4”), 68.2 (CH₂, C-5”), 111.8 (CH, C-8), 115.5 (CH, C-4), 116.3 (CH, d, J 21.9 Hz, C-3’), 120.8 (CH, C-6), 121.8 (C₀, C-4b), 121.9 (CH, C-5), 125.1 (CH, C-6’), 125.3 (C₀, C-4a), 129.0 (CH, C-7), 129.8 (C₀, C-9a), 130.9 (CH, d, J 7.9 Hz, C-4’), 132.7 (CH, C-5’), 135.2 (C₀, C-3), 136.8 (C₀, C-1’), 138.0 (C₀, C-8a), 140.6 (C₀, C-1), 159.9 (C₀, d, J 245 Hz, 1C, C-2’), 164.9 (C₀, C-2”); HRMS-ESI m/z, calcd. for C₂₀H₁₅FN₃O [M + H]⁺: 332.1194, found: 332.1166.

1-(4-Fluorophenyl)-3-(4,5-dihydro-1,3-oxazol-2-yl)-9H-β-carboline (8c)

Yield: 51%; mp > 246 °C (decomp.); ¹H NMR (300 MHz, CDCl₃) δ_H 4.15 (t, J 9.5 Hz, 2H, H-4”), 4.57 (t, 9.5 Hz, 2H, H-5”), 7.08 (t, J 8.8 Hz, 2H, H-3’, H-5’), 7.33 (ddd, J 7.9, 5.7, 2.3 Hz, 1H, H-6), 7.53-7.56 (m, 2H, H-7, H-8), 7.84 (dd, J 8.8, 5.4 Hz, 2H, H-2’, H-6’), 8.14 (d, J 7.9 Hz, 1H, H-5), 8.70 (s, 1H, H-4), 9.16 (s, 1H, NH, H-9); ¹³C NMR (75 MHz, CDCl₃) δ_C 54.9 (CH₂, C-4”), 68.2 (CH₂, C-5”), 111.9 (CH, C-8), 115.1 (CH, C-4), 115.9 (d, J 21.9 Hz, 2CH, C-3’, C-5’), 120.9 (CH, C-6), 121.9 (CH, C-5), 122.0 (C₀, C-4b), 128.9 (CH, C-7), 129.9 (C₀, C-4a), 130.2 (d, J 8.5 Hz, 2CH, C-2’, C-6’), 134.0 (C₀, C-9a), 134.3 (C₀, C-3), 136.6 (C₀, C-1’), 140.8 (C₀, C-8a), 141.9 (C₀, C-1), 163.1 (d, J 247.2 Hz, C₀, C-4’), 165.0 (C₀, C-2”); HRMS-ESI m/z, calcd. for C₂₀H₁₅FN₃O [M + H]⁺: 332.1194, found: 332.1149.

1-(2-Chlorophenyl)-3-(4,5-dihydro-1,3-oxazol-2-yl)-9H-β-carboline (8d)

Yield: 65%; mp 246-249 °C; ¹H NMR (500 MHz, CDCl₃/CD₃OD) δ_H 4.14 (t, J 9.6 Hz, 2H, H-4”), 4.58 (t, J 9.6 Hz, 2H, H-5”), 7.32 (m, 1H, H-6), 7.38-7.45 (m, 2H, H-7, H-6’), 7.51-7.61 (m, 4H, H-8, H-3’, H-4’, H-5’), 8.20 (d, J 7.9 Hz, 1H, H-5), 8.80 (s, 1H, H-4); ¹³C NMR (125 MHz, CDCl₃/CD₃OD) δ_C 54.5 (CH₂, C-4”), 68.4 (CH₂, C-5”), 112.1 (CH, C-8), 115.9 (CH, C-4), 120.7 (CH, C-6), 121.7 (C₀, C-4b), 122.0 (CH, C-5), 127.3 (CH, C-7), 129.0 (CH, C-5’), 129.4 (C₀, C-4a), 129.8 (CH, C-3’), 130.4 (CH, C-6’), 132.3 (CH, C-4’), 133.4 (C₀, C-2’), 135.4 (C₀, C-9a), 135.6 (C₀, C-3), 136.7 (C₀, C-1’), 141.2 (C₀, C-8a), 141.4 (C₀, C-1), 165.6 (C₀, C-2”); HRMS-ESI m/z, calcd. for C₂₀H₁₅ClN₃O [M + H]⁺: 348.0898, found: 348.0854.

1-(4-Chlorophenyl)-3-(4,5-dihydro-1,3-oxazol-2-yl)-9H-β-carboline (8e)

Yield: 50%; mp 278-282 °C; ¹H NMR (300 MHz, CDCl₃) δ_H 4.19 (t, J 9.5 Hz, 2H, H-4”), 4.59 (t, J 9.5 Hz, 2H, H-5”), 7.36 (t, J 7.9 Hz, 1H, H-6), 7.51-7.62 (m, 4H, H-7, H-8, H-3’, H-5’), 7.91 (d, J 8.2 Hz, 2H, H-2’, H-6’), 8.18 (d, J 7.9 Hz, 1H, H-5), 8.68 (s, 1H, NH, H-9), 8.75 (s, 1H, H-4); ¹³C NMR (75 MHz, CDCl₃) δ_C 55.1 (CH₂, C-4”), 68.3 (CH₂, C-5”), 111.8 (CH, C-8), 115.3 (CH, C-4), 121.1 (CH, C-6), 122.0 (CH, C-5), 122.0 (C₀, C-4b), 129.1 (CH, C-7), 129.4 (2CH, C-3’, C-5’), 129.7 (2CH, C-2’, C-6’), 130.2 (C₀, C-4a), 134.3 (C₀, C-4’), 135.1 (C₀, C-9a), 136.4 (C₀, C-3), 137.1 (C₀, C-1’), 140.6 (C₀, C-8a), 141.6 (C₀, C-1), 164.8 (C₀, C-2”); HRMS-ESI m/z, calcd. for C₂₀H₁₅ClN₃O [M + H]⁺: 348.0898, found: 348.0906.

1-(3-Nitrophenyl)-3-(4,5-dihydro-1,3-oxazol-2-yl)-9H-β-carboline (8f)

Yield: 58%; mp > 250 °C (decomp.); ¹H NMR (500 MHz, DMSO-d₆) δ_H 4.06 (t, J 9.5 Hz, 2H, H-4”), 4.50 (t, J 9.5 Hz, 2H, H-5”), 7.34 (t, J 7.9 Hz, 1H, H-6), 7.61-7.69 (m, 2H, H-7, H-8), 7.93 (t, J 7.9 Hz, 1H, H-5’), 8.39-8.44 (m, 2H, H-5, H-4’), 8.48 (d, J 7.9 Hz, 1H, H-6’), 8.79 (t, J 1.8 Hz, 1H, H-2’), 8.87 (s, 1H, H-4), 12.01 (s, 1H, NH, H-9); ¹³C NMR (125 MHz, DMSO-d₆) δ_C 54.4 (CH₂, C-4”), 67.4 (CH₂, C-5”), 112.5 (CH, C-8), 115.6 (CH, C-4), 120.3 (CH, C-6), 120.8 (C₀, C-4b), 122.1 (CH, C-5), 123.2 (CH, C-2’), 123.4 (CH, C-4’), 128.9 (CH, C-7), 129.9 (C₀, C-4a), 130.3 (CH, C-5’), 133.9 (C₀, C-9a), 134.8 (CH, C-6’), 135.9 (C₀, C-3), 139.0 (C₀, C-1’), 139.0 (C₀, C-8a), 141.5 (C₀, C-1), 148.1 (C₀, C-3’), 163.4 (C₀, C-2”); HRMS-ESI m/z, calcd. for C₂₀H₁₅N₄O₃ [M + H]⁺: 359.1139, found: 359.1098.

1-(4-Nitrophenyl)-3-(4,5-dihydro-1,3-oxazol-2-yl)-9H-β-carboline (8g)

Yield: 74%; mp 323-326 °C; ¹H NMR (300 MHz, DMSO-d₆) δ_H 4.06 (t, J 9.5 Hz, 2H, H-4”), 4.50 (t, J 9.5 Hz, 2H, H-5”), 7.34 (ddd, J 7.9, 7.0, 1.1 Hz, 1H, H-6), 7.63 (ddd, J 8.2, 7.0, 1.1 Hz, 1H, H-7), 7.69 (d, J 8.2 Hz, 1H, H-8), 8.32 (d, J 9.0 Hz, 2H, H-2’, H-6’), 8.43 (d, J 7.9 Hz, 1H, H-5), 8.47 (d, J 9.0 Hz, 2H, H-3’, H-5’), 8.88 (s, 1H, H-4), 12.01 (s, 1H, NH, H-9); ¹³C NMR (75 MHz, DMSO-d₆) δ_C 54.5 (CH₂, C-4”), 67.5 (CH₂, C-5”), 112.6 (CH, C-8), 115.9 (CH, C-4), 120.5 (CH, C-6), 120.9 (C₀, C-4b), 122.2 (CH, C-5), 123.9 (2CH, C-3’, C-5’), 129.0 (CH, C-7), 129.8 (2CH, C-2’, C-6’), 130.1 (C₀, C-4a), 134.1 (C₀, C-9a), 136.1 (C₀, C-3), 139.0 (C₀, C-1’), 141.6 (C₀, C-8a), 143.9 (C₀, C-1), 147.4 (C₀, C-4’), 163.5 (C₀, C-2”); HRMS-ESI m/z, calcd. for C₂₀H₁₅N₄O₃ [M + H]⁺: 359.1139, found: 359.1101.

1-(4-Methoxyphenyl)-3-(4,5-dihydro-1,3-oxazol-2-yl)-9H-β-carboline (8h)

Yield: 81%; mp 247-250 °C; ¹H NMR (500 MHz, CDCl₃) δ_H 3.84 (s, 3H, OCH₃), 4.16 (t, J 9.5 Hz, 2H, H-4”), 4.57 (t, J 9.5 Hz, 2H, H-5”), 6.98 (d, J 8.4 Hz, 2H, H-3’, H-5’), 7.33 (t, J 7.8 Hz, 1H, H-6), 7.52-7.57 (m, 2H, H-7, H-8), 7.86 (d, J 8.4 Hz, 2H, H-2’, H-6’), 8.15 (d, J 7.8 Hz, 1H, H-5), 8.69 (s, 1H, H-4), 8.95 (s, 1H, NH, H-9); ¹³C NMR (125 MHz, CDCl₃) δ_C 55.0 (CH₂, C-4”), 55.4 (OCH₃), 68.2 (CH₂, C-5”), 111.8 (CH, C-8), 114.4 (2CH, C-3’, C-5’), 114.6 (CH, C-4), 120.8 (CH, C-6), 121.9 (CH, C-5), 122.1 (C₀, C-4b), 128.7 (CH, C-7), 129.6 (2CH, C-2’, C-6’), 130.5 (C₀, C-4a), 134.3 (C₀, C-9a), 136.7 (C₀, C-3), 136.7 (C₀, C-1’), 140.7 (C₀, C-8a), 142.8 (C₀, C-1), 160.2 (C₀, C-4’), 165.1 (C₀, C-2”); HRMS-ESI m/z, calcd. for C₂₁H₁₈N₃O₂ [M + H]⁺: 344.1394, found: 344.1396.

1-(4-(Dimethylamino)phenyl)-3-(4,5-dihydro-1,3-oxazol-2-yl)-9H-β-carboline (8i)

Yield: 93%; mp 235-238 °C; ¹H NMR (500 MHz, CDCl₃) δ_H 2.96 (s, 6H, N(CH₃)₂), 4.16 (t, J 9.5 Hz, 2H, H-4”), 4.56 (t, J 9.5 Hz, 2H, H-5”), 6.72 (d, J 8.7 Hz, 2H, H-3’, H-5’), 7.30 (dt, J 8.0, 4.0 Hz, 1H, H-6), 7.52-7.53 (m, 2H, H-7, H-8), 7.81 (d, J 8.7 Hz, 2H, H-2’, H-6’), 8.13 (d, J 8.0 Hz, 1H, H-5), 8.63 (s, 1H, H-4), 9.03 (s, 1H, NH, H-9); ¹³C NMR (125 MHz, CDCl₃) δ_C 40.3 (N(CH₃)₂), 55.0 (CH₂, C-4”), 68.1 (CH₂, C-5”), 111.8 (CH, C-8), 112.3 (2CH, C-3’, C-5’), 113.9 (CH, C-4), 120.5 (CH, C-6), 121.8 (CH, C-5), 122.2 (C₀, C-4b), 125.7 (C₀, C-4a), 128.4 (CH, C-7), 129.2 (2CH, C-2’, C-6’), 129.3 (C₀, C-9a), 134.3 (C₀, C-3), 136.5 (C₀, C-1’), 140.6 (C₀, C-8a), 143.6 (C₀, C-1), 150.8 (C₀, C-4’), 165.3 (C₀, C-2”); HRMS-ESI m/z, calcd. for C₂₂H₂₁N₄O [M + H]⁺: 357.1710, found: 357.1712.

Synthesis of 1-(substituted-phenyl)-3-(5,6-dihydro-4H-1,3-oxazin-2-yl)-β-carboline (9a-9h)

To a solution of intermediates 3-chloropropyl-β-carboline-3-carboxamides 7a-7h (0.3 mmol) in DMF (3 mL), it was added 2 equivalents of potassium carbonate (0.08 g). The reaction mixture was irradiated in a microwave oven at 100% power level for 5-9 min. After all the starting material was consumed (5-9 min), the reaction mixture was poured into water and the precipitate formed was filtered under vacuum, washed with water and recrystallized with acetonitrile. The derivatives 9a-9h were obtained in yields in the range of 45-86%.

1-Phenyl-3-(5,6-dihydro-4H-1,3-oxazin-2-yl)-9H-β-carboline (9a)

Yield: 45%; mp 231-234 °C; ¹H NMR (300 MHz, CDCl₃/CD₃OD) δ_H 2.04 (qt, J 5.4 Hz, 2H, H-5”), 3.68 (t, J 5.4 Hz, 2H, H-4”), 4.47 (t, J 5.4 Hz, 2H, H-6”), 7.25-7.31 (m, 1H, H-6), 7.36-7.54 (m, 5H, H-7, H-8, H-3’, H-4’, H-5’), 7.91 (d, J 7.0 Hz, 2H, H-2’, H-6’), 8.14 (d, J 7.8 Hz, 1H, H-5), 8.61 (s, 1H, H-4); ¹³C NMR (75 MHz, CDCl₃/CD₃OD) δ_C 21.9 (CH₂, C-5”), 42.8 (CH₂, C-4”), 65.6 (CH₂, C-6”), 111.8 (CH, C-8), 113.4 (CH, C-4), 120.4 (CH, C-6), 121.9 (CH, C-5), 122.1 (C₀, C-4b), 128.5 (2CH, C-2’, C-6’), 128.5 (CH, C-4’), 128.8 (CH, C-7), 128.9 (CH, C-3’, C-5’), 130.0 (C₀, C-4a), 134.3 (C₀, C-9a), 138.3 (C₀, C-1’), 140.9 (C₀, C-8a), 141.6 (C₀, C-3), 142.3 (C₀, C-1), 156.3 (C₀, C-2”); HRMS-ESI m/z, calcd. for C₂₁H₁₈N₃O [M + H]⁺: 328.1444, found: 328.1476.

1-(2-Fluorophenyl)-3-(5,6-dihydro-4H-1,3-oxazin-2-yl)-9H-β-carboline (9b)

Yield: 73%; mp 234-239 °C; ¹H NMR (300 MHz, CDCl₃) δ_H 2.07 (qt, J 5.6 Hz, 2H, H-5”), 3.74 (t, J 5.6 Hz, 2H, H-4”), 4.51 (t, J 5.6 Hz, 2H, H-6”), 7.21-7.35 (m, 3H, H-6, H-3’, H-6’), 7.43-7.59 (m, 3H, H-4’, H-7, H-8), 7.91 (td, J 7.5, 1.6 Hz, 1H, H-5’), 8.19 (d, J 7.8 Hz, 1H, H-5), 8.46 (s, 1H, NH, H-9), 8.74 (s, 1H, H-4); ¹³C NMR (75 MHz, CDCl₃) δ_C 22.0 (CH₂, C-5”), 43.0 (CH₂, C-4”), 65.7 (CH₂, C-6”), 111.7 (CH, C-8), 113.9 (CH, C-4), 115.8 (CH, d, J 21.9 Hz, C-3’), 120.6 (CH, C-6), 122.0 (CH, C-5), 122.1 (C₀, C-4b), 125.1 (CH, C-6’), 125.2 (C₀, C-4a), 128.8 (CH, C-7), 130.1 (C₀, C-9a), 130.7 (CH, d, J 8.5 Hz, C-4’), 132.9 (CH, d, J 4.0 Hz, C-5’), 135.0 (C₀, C-1’), 137.2 (C₀, C-8a), 140.7 (C₀, C-3), 142.2 (C₀, C-1), 155.7 (C₀, C-2”), 160.0 (C₀, d, J 244.9 Hz, 1C, C-2’); HRMS-ESI m/z, calcd. for C₂₁H₁₇FN₃O [M + H]⁺: 346.1350, found: 346.1345.

1-(4-Fluorophenyl)-3-(5,6-dihydro-4H-1,3-oxazin-2-yl)-9H-β-carboline (9c)

Yield: 45%; mp 228-231 °C; ¹H NMR (300 MHz, CDCl₃/CD₃OD) δ_H 2.06 (dt, J 5.7 Hz, 2H, H-5”), 3.70 (t, J 5.7 Hz, 2H, H-4”), 4.49 (t, J 5.7 Hz, 2H, H-6”), 7.14 (t, J 8.7 Hz, 2H, H-3’, H-5’), 7.28-7.33 (m, 1H, H-6), 7.49-7.57 (m, 2H, H-7, H-8), 7.91 (dd, J 8.7, 5.5 Hz, 2H, H-2’, H-6’), 8.15 (d, J 7.9 Hz, 1H, H-5), 8.57 (s, 1H, H-4); ¹³C NMR (75 MHz, CDCl₃/CD₃OD) δ_C 21.9 (CH₂, C-5”), 42.7 (CH₂, C-4”), 65.6 (CH₂, C-6”), 111.9 (CH, C-8), 113.4 (CH, C-4), 115.8 (d, J 21.3 Hz, 2CH, C-3’, C-5’), 120.5 (CH, C-6), 121.8 (CH, C-5), 122.1 (C₀, C-4b), 128.6 (CH, C-7), 130.1 (C₀, C-4a), 130.4 (d, J 8.5 Hz, 2CH, C-2’, C-6’), 134.1 (C₀, C-9a), 134.4 (C₀, C-1’), 141.0 (C₀, C-8a), 141.2 (C₀, C-3), 141.4 (C₀, C-1), 156.4 (C₀, C-2”), 163.1 (d, J 246.6 Hz, C₀, C-4’); HRMS-ESI m/z, calcd. for C₂₁H₁₇FN₃O [M + H]⁺: 346.1350, found: 346.1386.

1-(2-Chlorophenyl)-3-(5,6-dihydro-4H-1,3-oxazin-2-yl)-9H-β-carboline (9d)

Yield: 86%; mp 236-238 °C; ¹H NMR (300 MHz, DMSO-d₆) δ_H 1.93 (qt, J 5.5 Hz, 2H, H-5”), 3.55 (t, J 5.5 Hz, 2H, H-4”), 4.36 (t, J 5.5 Hz, 2H, H-6”), 7.27 (dt, J 7.8, 4.0 Hz, 1H, H-6), 7.51-7.70 (m, 6H, H-7, H-8, H-3’, H-4’, H-5’, H-6’), 8.35 (d, J 7.8 Hz, 1H, H-5), 8.75 (s, 1H, H-4), 11.48 (s, 1H, NH, H-9); ¹³C NMR (75 MHz, DMSO-d₆) δ_C 21.6 (CH₂, C-5”), 42.1 (CH₂, C-4”), 64.7 (CH₂, C-6”), 112.2 (CH, C-8), 113.6 (CH, C-4), 119.8 (CH, C-6), 121.1 (C₀, C-4b), 122.0 (CH, C-5), 127.4 (CH, C-7), 128.5 (CH, C-5’), 128.6 (C₀, C-4a), 129.6 (CH, C-3’), 130.4 (CH, C-6’), 132.0 (CH, C-4’), 132.6 (C₀, C-2’), 134.5 (C₀, C-9a), 136.9 (C₀, C-1’), 140.0 (C₀, C-8a), 140.7 (C₀, C-3), 141.4 (C₀, C-1), 154.7 (C₀, C-2”); HRMS-ESI m/z, calcd. for C₂₁H₁₇ClN₃O [M + H]⁺: 362.1055, found: 362.1036.

1-(4-Chlorophenyl)-3-(5,6-dihydro-4H-1,3-oxazine)-9H-β-carboline (9e)

Yield: 60%; mp 234-236 °C; ¹H NMR (300 MHz, DMSO-d₆) δ_H 1.95 (qt, J 5.4 Hz, 2H, H-5”), 3.57 (t, J 5.4 Hz, 2H, H-4”), 4.40 (t, J 5.4 Hz, 2H, H-6”), 7.28 (t, J 7.9 Hz, 1H, H-6), 7.55-7.63 (m, 2H, H-7, H-8), 7.68 (d, J 8.4 Hz, 2H, H-3’, H-5’), 8.04 (d, J 8.4 Hz, 2H, H-2’, H-6’), 8.35 (d, J 7.9 Hz, 1H, H-5), 8.70 (s, 1H, H-4), 11.69 (s, 1H, NH, H-9); ¹³C NMR (75 MHz, DMSO-d₆) δ_C 21.6 (CH₂, C-5”), 42.1 (CH₂, C-4”), 64.8 (CH₂, C-6”), 112.5 (CH, C-8), 113.4 (CH, C-4), 120.0 (CH, C-6), 121.2 (CH, C-4b), 121.9 (C₀, C-5), 128.5 (CH, C-7), 128.7 (2CH, C-3’, C-5’), 129.8 (C₀, C-4a), 130.3 (2CH, C-2’, C-6’), 133.4 (C₀, C-4’), 133.5 (C₀, C-9a), 136.8 (C₀, C-1’), 139.4 (C₀, C-8a), 141.2 (C₀, C-3), 141.6 (C₀, C-1), 154.7 (C₀, C-2”); HRMS-ESI m/z, calcd. for C₂₁H₁₇ClN₃O [M + H]⁺: 362.1055, found: 362.1046.

1-(3-Nitrophenyl)-3-(5,6-dihydro-4H-1,3-oxazin-2-yl)-9H-β-carboline (9f)

Yield: 51%; mp 255-258 °C; ¹H NMR (300 MHz, DMSO-d₆) δ_H 1.96 (qt, J 5.5 Hz, 2H, H-5”), 3.59 (t, J 5.5 Hz, 2H, H-4”), 4.42 (t, J 5.5 Hz, 2H, H-6”), 7.31 (t, J 7.2 Hz, 1H, H-6), 7.57-7.67 (m, 2H, H-7, H-8), 7.91 (t, J 7.6 Hz, 1H, H-5’), 8.37-8.40 (m, 2H, H-4’, H-5), 8.45 (d, J 7.6 Hz, 1H, H-6’), 8.76-8.77 (m, 2H, H-2’, H-4), 11.86 (s, 1H, NH, H-9); ¹³C NMR (75 MHz, DMSO-d₆) δ_C 21.6 (CH₂, C-5”), 42.1 (CH₂, C-4”), 64.9 (CH₂, C-6”), 112.5 (CH, C-8), 114.0 (CH, C-4), 120.3 (CH, C-6), 121.2 (C₀, C-4b), 122.1 (CH, C-5), 123.2 (CH, C-2’), 123.3 (CH, C-4’), 128.8 (CH, C-7), 130.2 (C₀, C-4a), 130.4 (CH, C-5’), 133.8 (C₀, C-9a), 135.0 (CH, C-6’), 138.3 (C₀, C-1’), 139.5 (C₀, C-8a), 141.4 (C₀, C-3), 141.7 (C₀, C-1), 148.2 (C₀, C-3’), 154.6 (C₀, C-2”); HRMS-ESI m/z, calcd. for C₂₁H₁₇N₄O₃ [M + H]⁺: 373.1295, found: 373.1295.

1-(4-Nitrophenyl)-3-(5,6-dihydro-4H-1,3-oxazin-2-yl)-9H-β-carboline (9g)

Yield: 51%; mp 297-300 °C; ¹H NMR (300 MHz, DMSO-d₆) δ_H 1.96 (qt, J 5.5 Hz, 2H, H-5”), 3.59 (t, J 5.5 Hz, 2H, H-4”), 4.41 (t, J 5.5 Hz, 2H, H-6”), 7.31 (t, J 7.8 Hz, 1H, H-6), 7.58-7.68 (m, 2H, H-7, H-8), 8.30 (d, J 9.0 Hz, 2H, H-3’, H-5’), 8.39 (d, J 7.8 Hz, 1H, H-5), 8.46 (d, J 9.0 Hz, 2H, H-2’, H-6’), 8.79 (s, 1H, H-4), 11.87 (s, 1H, NH, H-9); ¹³C NMR (75 MHz, DMSO-d₆) δ_C 21.6 (CH₂, C-5”), 42.1 (CH₂, C-4”), 64.9 (CH₂, C-6”), 112.5 (CH, C-8), 114.3 (CH, C-4), 120.3 (CH, C-6), 121.1 (C₀, C-4b), 122.0 (CH, C-5), 123.9 (2CH, C-2’, C-6’), 128.9 (CH, C-7), 129.8 (2CH, C-3’, C-5’), 130.3 (C₀, C-4a), 133.9 (C₀, C-9a), 138.1 (C₀, C-1’), 141.4 (C₀, C-8a), 141.7 (C₀, C-3), 144.3 (C₀, C-1), 147.3 (C₀, C-4’); HRMS-ESI m/z, calcd. for C₂₁H₁₇N₄O₃ [M + H]⁺: 373.1295, found: 373.1281.

1-(4-Methoxyphenyl)-3-(5,6-dihydro-4H-1,3-oxazin-2-yl)-9H-β-carboline (9h)

Yield: 66%; mp 219-221 °C; ¹H NMR (500 MHz, CDCl₃) δ_H 2.05 (qt, J 5.6 Hz, 2H, H-5”), 3.71 (t, J 5.6 Hz, 2H, H-4”), 3.83 (s, 3H, OCH₃), 4.48 (t, J 5.6 Hz, 2H, H-6”), 6.96 (d, J 8.2 Hz, 2H, H-3’, H-5’), 7.30 (t, J 7.8 Hz, 1H, H-6), 7.49-7.54 (m, 2H, H-7, H-8), 7.85 (d, J 8.2 Hz, 2H, H-2’, H-6’), 8.15 (d, J 7.8 Hz, 1H, H-5), 8.63 (s, 1H, H-4), 8.89 (s, 1H, NH, H-9); ¹³C NMR (125 MHz, CDCl₃) δ_C 22.0 (CH₂, C-5”), 42.9 (CH₂, C-4”), 55.4 (OCH₃), 65.6 (CH₂, C-6”), 111.7 (CH, C-8), 112.9 (CH, C-4), 114.3 (2CH, C-3’, C-5’), 120.5 (CH, C-6), 121.9 (CH, C-5), 122.3 (C₀, C-4b), 128.4 (CH, C-7), 129.7 (2CH, C-2’, C-6’), 129.9 (C₀, C-4a), 130.9 (C₀, C-9a), 134.1 (C₀, C-3), 140.7 (C₀, C-1’), 141.9 (C₀, C-8a), 142.1 (C₀, C-1), 156.0 (C₀, C-4’), 160.1 (C₀, C-2”); HRMS-ESI m/z, calcd. for C₂₂H₂₀N₃O₂ [M + H]⁺: 358.1550, found: 358.1567.

Antileishmanial activity

Parasites and cell culture

Leishmania amazonensis promastigotes were maintained at 25 °C in Warren medium supplemented with 10% FBS (fetal bovine serum). J774-A1 macrophages were maintained at 37 °C under 5% CO₂ atmosphere in Roswell Park Memorial Institute (RPMI) 1640 medium (pH 7.2) supplemented with 10% FBS.

Antiprotozoal activity

The effects of synthesized compounds were evaluated in promastigotes of L. amazonensis in log phase of growth (48 h) at concentration of 1 × 10⁻⁶ cells mL⁻¹. The promastigotes were added into sterile 96-well microplates containing increasing concentrations of compounds 6a-6i and 7a-7i, 8a-8i and 9a-9h. After incubation for 72 h at 25 °C, it was added 50 µL of solution of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide/phenazine methosulfate (XTT/PMS) (XTT/PMS, 0.5 and 0.3 mg mL⁻¹) in the absence of light. After 4 h, the absorbance was read in a spectrophotometer at 450 nm. The IC₅₀ values (concentration that inhibit 50% of cells) was determined by linear regression. Miltefosine was used as positive control.

The effects of compounds 6d, 8d, 8i, 9a, 9e and 9h were also evaluated in intracellular amastigotes, in this antiproliferative assay, J774A1 macrophages (5 × 10⁵cells mL⁻¹) and promastigotes (5 × 10⁶parasites mL⁻¹) were added in a plate with coverslips and incubated at 34 °C with 5% CO₂ during 24 h. The treatment was performed after 24 h with compounds in increasing concentrations and incubated for 48 h. For the determination of IC₅₀, the glass coverslips were fixed and stained with Panótico kit as indicated by the manufacturer and 200 macrophages per coverslip were evaluated on a light microscope. The number of macrophages infected, the number of amastigotes within each infected macrophage and the survival index (infected cells percentage × amastigote average per infected macrophage) were determined. Survival index of amastigotes from untreated infected macrophages was considered as 100% of survival.

Cytotoxicity assay

The cytotoxicity was evaluated in J774-A1 macrophages. The macrophages at concentration of 5 × 10⁻⁵ cells mL⁻¹ in RPMI 1640 medium supplemented with 10% FBS were introduced into sterile 96-well micro plates and incubated for 24 h at 37 °C and 5% of CO₂ tension. After this period, the supernatant was removed and increasing concentrations of the substances were added. After 48 h of incubation under the same conditions mentioned above, the cells were washed with 0.01 M PBS (phosphate-buffered saline) and 50 µL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (2 mg mL⁻¹) was added to each well and incubated at absence of light at 34 °C. After 4 h, 150 µL of DMSO was added in order to solubilize formazan crystals. The absorbance was read at 570 nm in microplate reader (Biotek Power Wave XS spectrofluorometer). The concentration that decreased 50% (CC₅₀) of viability of macrophages was determined by linear regression analysis of the data.

Statistical analysis

The data shown in the tables are expressed as the mean ± standard deviation of at least three independent experiments. The statistical analysis was performed using GraphPad Prism 6.0 software.⁴⁰ The samples were analyzed using one-way analysis of variance (ANOVA), and the Tukey post hoc test was used to compare means when appropriate. Values of p ≤ 0.05 were considered statistically significant.

Spin labeling and EPR spectroscopy

Promastigotes of L. amazonensis in suspension at 5 × 10⁷parasites mL⁻¹ (2 mL) were incubated for 2 h at 26 °C in culture medium without fetal calf serum (FCS) and containing 150 µM of the treatment compound. After incubation, the sample was centrifuged at 1800 × g for 10 min to increase the cell concentration to 1 × 10⁸parasites mL⁻¹ and decrease the final volume to 50 µL. To incorporate the spin label 5-DSA into the parasite membrane first a spin label film was made on the bottom of a glass tube. An aliquot (1 µL) of a 5-DSA ethanolic solution (4 mg mL⁻¹) was added to the tube and after evaporation of ethanol the parasite suspension was placed on the film and stirred gently. Then, the sample was transferred to a 1-mm-i.d. (internal diameter) capillary tube for the EPR measurements.

Spectra were recorded using an EPR EMX-Plus spectrometer of Bruker (Rheinstetten, Germany) with the following spectrometer settings: modulation frequency, 100 kHz; modulation amplitude, 1.0 G; microwave power, 2 mW; magnetic field scan, 100 G; and sample temperature, 25 °C.

Supplementary Information

Supplementary information (HRMS, ¹H and ¹³C NMR spectral data) is available free of charge at http://jbcs.sbq.org.br as PDF file.

Acknowledgments

This work was supported by Fundação Araucária, PR, Brazil (M. H. S., No. 2/2017 Prot. 47223 FA/UEM) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ, A. A., 303829/2016-8). We thank CAPES and CNPq for doctoral (P. B.) and postdoctoral (L. A., 150369/2018-2) fellowships, and to Complexo de Centrais de Apoio à Pesquisa of Universidade Estadual de Maringá (COMCAP-UEM) for the facilities.

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Publication Dates

• Publication in this collection
  08 June 2020
• Date of issue
  June 2020

History

• Received
  17 Oct 2019
• Accepted
  10 Jan 2020