Pharmacochemical Studies of Synthesized Coumarin–Isoxazole–Pyridine Hybrids

Abstract

Several new coumarin–isoxazole–pyridine hybrids were synthesized through a 1,3-dipolar cycloaddition reaction of nitrile oxides, prepared in situ from pyridine aldehyde oximes, with propargyloxy- or propargylaminocoumarins in moderate-to-good yields. Synthetic modifications were applied using (diacetoxyiodo)benzene (PIDA) at room temperature, microwave irradiation, or tert-butyl nitrite (TBN) under reflux. Coumarin, isoxazole, and pyridine groups were selected for hybridization in one molecule due to their biological impact to inhibit lipid peroxidation and an enzyme implicated in inflammation. Preliminary in vitro screening tests for lipoxygenase (LOX) inhibition and anti-lipid peroxidation for the new hybrids were performed. A discussion on the structure–activity relationship is presented. Compounds 12b and 13a were found to be potent LOX inhibitors with IC₅₀ 5 μΜ and 10 μΜ, respectively, while 12b presented high (90.4%) anti-lipid peroxidation. Furthermore, hybrids 12b and 13a exhibited moderate-to-low anticancer activities on HeLa, HT-29, and H1437 cancer cells.

1. Introduction

Reactive oxygen species (ROS) are continuously produced in the human body as byproducts of cell metabolism. Some of them are characterized as highly toxic [1]. Their extreme reactivity and the tendency to induce chain reactions lead to pathological processes like inflammation, asthma, and cardiovascular and neurological disorders. Lipid peroxidation is one of the major outcomes of ROS-mediated injury. It directly damages membranes and generates several products that possess neurotoxic activity [1]. ROS exerts toxic effects and directly oxidizes biological macromolecules, such as proteins, nucleic acids, and lipids, further exacerbating the development of inflammatory responses and causing various inflammatory diseases.

Lipoxygenase (LOX) is the key enzyme in leukotriene biosynthesis [2]. Leukotrienes are derived from the biotransformation of arachidonic acid catalyzed by 5-lipoxygenase (5-LOX). They are inflammatory mediators, causing inflammation, cancer, and stroke. LOXs contribute to membrane lipid peroxidation by forming hydroperoxides in the lipid bilayer, whereas cerebral ischemia-reperfusion triggers lipid peroxidation and inflammation. Inhibitors of LOX have attracted attention initially as potential agents for inflammatory disease treatment and for certain types of cardiovascular diseases [2].

Coumarin derivatives of natural or synthetic origin represent a large variety of compounds with diverse biological and pharmacological properties [3,4,5,6,7,8,9,10]. These properties include, amongst others, anti-HIV [11], anticancer [12], antioxidant [13], anti-inflammatory [13,14], anti-Alzheimer [15], antidepressant [16], antibacterial [17], anticonvulsant [18], antitubercular [19], and anticoagulant [20] activities.

The hybrid drug concept is an alternative sophisticated approach of combination therapy applicable in the treatment of complex and multifactorial diseases such as cancer, infectious and inflammatory diseases, and neurological disorders when traditional single-target therapy is not satisfactory [21]. Hybrids that scavenge ROS have emerged as an important approach used to limit inflammatory responses and protect the host against damage [22]. Molecular hybridization is a drug design strategy that combines two or more pharmacophore groups into a single multi-functional molecule [23,24]. In the last decade, coumarin-isoxazole hybrids, among coumarin derivatives, have been synthesized, as they offer diverse biological activities, such as antibacterial [25,26], anticancer [27,28], antiviral, anti-inflammatory, anti-psychotic, antidiabetic [28], antiproliferative [29], antimicrobial [30,31], anticoagulant, and anticholinesterase activities [32]. Hybrids containing coumarin–pyridine scaffold also exhibit a plethora of biological activities, such as anticancer [33,34,35], anti-Alzheimer, antitubercular, antimicrobial, antiviral [36], anti-osteoporotic [37], and antileishmanial activities [38]. Additionally, isoxazole-pyridine hybrids present interesting biological properties, such as anti-acetylcholinesterase [39], anticancer, antioxidant [40], antitubercular [41], and inhibition of human cytochrome P-450 2A6 [42] activities.

An important method for the synthesis of isoxazole derivatives is the Huisgen 1,3-dipolar cycloaddition reaction of nitrile oxides to alkynes, leading to the formation of 3,5-disubstituted isoxazoles [43,44]. The nitrile oxides are formed in situ from the corresponding aldoximes through chlorination and subsequent elimination of HCl by a base or oxidation of aldoxime using an oxidant [40,42]. (Diacetoxyiodo)benzene (PIDA) in room temperature (r.t.) [45], under heating [42], or under microwave irradiation [46] has been utilized for this oxidation. Other analogous reactions use hypochlorous acid at r.t. [39], cerum (IV) ammonium nitrate (CAN) under sonication [41], oxone at r.t. [41], tert-butyl nitrite (TBN) under heating [41], or (bis(trifluoroacetoxy)iodo)benzene (PIFA) under heating [39] as oxidants.

The available literature suggests that coumarin hybrids containing both coumarin with isoxazole and pyridine moieties do not exist. According to our knowledge, there is little evidence of coumarin hybrids with piperidine, dihydropyridine, or tetrahydropyridine framework. Piperidine hybrids display anti-filovirus [28,47], anti-psychotic [28,48], anti-acetylcholinesterase, and anti-butyrycholinesterase [22,49] activities. 1,4-Dihydropyridine hybrids present antidiabetic activity [28,50]. Previously, 1,2,3,4-tetrahydropyridine-fused coumarin with isooxazoline hybrid has been synthesized via an intramolecular 1,3-dipolar cycloaddition reaction [51]. Herein, in continuation of our ongoing interest in the synthesis and biological evaluation of coumarin hybrids [52,53,54,55], coumarin, isoxazole, and pyridine moieties were selected for hybridization in one molecule to investigate their biological capacity to inhibit lipid peroxidation and particularly enzymes like lipoxygenase (LOX), which are implicated in inflammation. Preliminary in vitro screening tests for LOX inhibition and anti-lipid peroxidation for the new hybrids were performed [56], and a discussion on the structure–activity relationship is presented. The synthesis of the hybrids was achieved by 1,3-dipolar cycloaddition reaction of pyridine aldoximes with propargyloxy- or propargylaminocoumarins. The studied reactions and the isolated products are depicted in Scheme 1, Scheme 2 and Scheme 3.

2. Results and Discussion

The 1,3-dipolar cycloaddition reaction of nitrile oxide, generated in situ from picolinaldehyde oxime (2) [57], with 1.1 equivalents of 7-propargyloxycoumarin (1a) [58], was selected as a model reaction for the investigation of suitable reaction conditions (Scheme 1). At first, PIDA, an efficient and inexpensive oxidizing reagent [45], was utilized for the in situ synthesis of the corresponding pyridine nitrile oxide. The reaction was performed using 1.1 equivalents of PIDA as the oxidant in methanol with a 0.057 M concentration of oxime at room temperature (Method A) to give the new 3,5-disubstituted isoxazole derivative 3a in 60% yield. The dimerization product of nitrile oxide, furoxan 4 [59] (20%), was also isolated from the reaction mixture (

Table 1. 1,3-Dipolar cycloaddition reactions of pyridine nitrile oxides with 7-propargyloxycoumarins.

Entry	Oxime	7-Propagyl Oxycoumarin	Method 1	Temperature	Time	Products (% Yield)
1	2	1a	A	r.t.	1 h	3a (60), 4 (20)
2	2	1a	B	120 °C	1 h	3a (48), 4 (16), 5 (9)
3	2	1a	C	Reflux	18 h	3a (34), 5 (32)
4	2	1b	A	r.t.	15 h	3b (65), 4 (17)
5	2	1b	B	120 °C	1 h	3b (43), 4 (17), 5 (11)
6	2	1b	C	Reflux	18 h	3b (44), 4 (15), 5 (13)
7	6	1a	A 2	r.t.	18 h	7a (24)
8	6	1a	C	Reflux	18 h	7a (61)
9	6	1b	A 2	r.t.	18 h	7b (30)
10	6	1b	C	Reflux	18 h	7b (53)
11	8	1a	D	Reflux	2 d	9a (24)
12	8	1a	C	Reflux	18 h	9a (42)
13	8	1b	D	Reflux	2 d	9b (12)
14	8	1b	C	Reflux	18 h	9b (45)

¹ Methods A, B, C, and D are referred to in Scheme 1 and Scheme 2 captions. ² Concentration of 0.015 M in MeOH.

, entry 1). Almost the same results were observed using a more diluted solution of oxime in a concentration of 0.015 M. HSQC experiments revealed the regiochemistry of 3a. The 4-H of the isoxazole ring at 7.04 ppm corresponds to 103.1 ppm, as depicted in HSQC, both characteristic of 4-H and 4-C of 3,5-diaryl-substituted isoxazoles [60]. Subsequently, we assessed if the use of microwave (MW) irradiation would affect the reaction’s results. The reaction in ethanol under MW at 120 °C for 1 h (Method B) resulted in isoxazole 3a in 48% yield, followed by furoxan 4 (16%) and 1,2,4-oxadiazole 5 [61] (9%) (Table 1, entry 2). Compound 5 was possibly formed by the 1,3-dipolar cycloaddition reaction of the nitrile oxide, with the corresponding nitrile obtained by dehydration of aldoxime 2 under the reaction conditions. The dehydration of aldoximes is a convenient route for the synthesis of nitriles under various conditions with a plethora of reagents [62]. A simple procedure, for example, is the transformation of nicotine aldehyde oxime to nicotine nitrile by heating in DMF as solvent at 135 °C [63]. As a third method, we selected TBN, a novel and green oxidizing reagent [41,64], as the oxidant for this reaction with acetonitrile as solvent under reflux for 18 h (Method C). The latter yielded the worst results with isoxazole 3a (34%) and 1,2,4-oxadiazole 5 (32%) isolated from the reaction mixture (Table 1, entry 3). It was evident that the increase in temperature favors the dehydration of oximes and the formation of 1,2,4-oxadiazole. The similar reactions of oxime 2 with 4-methyl-7-propargyloxycoumarin (1b) [58] under Methods A, B, or C resulted in the synthesis and isolation of isoxazole 3b in 65%, 43%, or 44% yield, respectively, along with furoxan 4 and 1,2,4-oxadiazole 5 (Table 1, entries 4–6). The above results indicate that in the case of picoline aldehyde oximes, Method A gave the better results.

Scheme 1. Reaction conditions: (i) Method A: propargyl coumarin (1.1 equiv.), PIDA (1.1 equiv.), oxime (1 equiv.) 0.057 M in MeOH; (ii) Method B: propargyl coumarin (1.1 equiv.), PIDA (1.1 equiv.), oxime (1 equiv.), EtOH, MW; (iii) Method C: propargyl coumarin (1.1 equiv.), TBN (1.1 equiv.), oxime (1 equiv.), MeCN.

Scheme 1. Reaction conditions: (i) Method A: propargyl coumarin (1.1 equiv.), PIDA (1.1 equiv.), oxime (1 equiv.) 0.057 M in MeOH; (ii) Method B: propargyl coumarin (1.1 equiv.), PIDA (1.1 equiv.), oxime (1 equiv.), EtOH, MW; (iii) Method C: propargyl coumarin (1.1 equiv.), TBN (1.1 equiv.), oxime (1 equiv.), MeCN.

We utilized, then, the nitrile oxide generated from nicotine aldehyde oxime (6) [42] in the reactions with 7-propargylocoumarins 1a and 1b (Scheme 2). The reactions of 1a and 1b under Method Ain a concentration of 0.015 M in MeOH afforded isoxazoles 7a and 7b in only 24% and 30% yields, respectively (Table 1, entry 7,9). The best results were under Method Cand led to the synthesis of isoxazoles 7a and 7b in 61% and 53% yields, respectively (Table 1, entries 8,10). The yield of these reactions is within the range previously reported for this nitrile oxide [42].

The reaction of nitrile oxide prepared from isonicotine aldehyde oxime (8) [65] with 7-propargyloxycoumarin (1a) was tested next under Method Abut had no results. When trifluoroacetic acid (TFA) (0.5 equiv.) was added with an oxime concentration of 0.015 M in EtOH (Method D), under reflux for 2 days, isoxazole 9a was isolated from the reaction mixture in 24% yield (Table 1, entry 11). The same reaction using Method C led to isoxazole 9a in 42% yield (Table 1, entry 12). The analogous reaction of oxime 8 with coumarin 1b under Method D led to isoxazole 9b in only 12% yield (Table 1, entry 13), while underMethod C, isoxazole 9b was isolated in 45% yield (Table 1, entry 14). The above results suggested that Method C is better with the nitrile oxides formed from nicotine aldehyde oxime (6) and isonicotine aldehyde oxime (8).

Scheme 2. Reaction conditions: (i) Method C; (ii) Method A with oxime 0.015 M in MeOH; (iii) Method D: propargyl coumarin (1.1 equiv.), PIDA (1.1 equiv.), oxime (1 equiv.) 0.015 M in EtOH and TFA (0.5 equiv.).

Scheme 2. Reaction conditions: (i) Method C; (ii) Method A with oxime 0.015 M in MeOH; (iii) Method D: propargyl coumarin (1.1 equiv.), PIDA (1.1 equiv.), oxime (1 equiv.) 0.015 M in EtOH and TFA (0.5 equiv.).

After the examination of 7-propargyloxycoumarins, we tested 1,3-dipolar cycloaddition reactions of 4-propargyloxycoumarin (10a) [66] and 4-propargylaminocoumarin (10b) [67] with the nitrile oxides formed from pyridine aldoximes (Scheme 3,

Table 2. 1,3-Dipolar cycloaddition reactions of pyridine nitrile oxides with 4-propargylcoumarins.

Entry	Oxime	4-Propargylcoumarin	Method 1	Temperature	Time	Products (% Yield)
1	2	10a	B	100 °C	1 h	11a (44), 4 (27)
2	2	10a	C	Reflux	18 h	11a (62), 4 (5), 5 (11)
3	2	10b	B	100 °C	2 h	11b (55)
4	6	10a	D	Reflux	2 d	12a (30)
5	6	10a	C	Reflux	18 h	12a (33)
6	6	10b	C	Reflux	18 h	12b (56)
7	8	10a	D	Reflux	2 d	13a (55)
8	8	10a	C	Reflux	18 h	13a (40)

¹ Methods B, C, and D are referred to in Scheme 1 and Scheme 2 captions.

). The reaction of 10a with 2 under Method B at 100 °C afforded isoxazole 11a (44%) and furoxan 4 (27%) (Table 2, entry 1), whereas under Method C, isoxazole 11a was synthesized in better yield (62%) accompanied by furoxan 4 (5%) and oxadiazole 5 (11%) (Table 2, entry 2). The similar reaction of 10b with oxime 2 led to isoxazole 11b (55%) under Method B (Table 2, entry 3). The analogous reaction of nicotine aldoxime (6) with 10a gave similar results under Method D and Method C, resulting in the isolation of isoxazole 12a in 30% and 33% yield, respectively (Table 2, entries 4,5). Oxime 6 reacted, also, with 4-propargylaminocoumarin (10b) under Method C to give isoxazole 12b in 56% yield (Table 2, entry 6). Isonicotine aldehyde oxime (8) was examined next for the reaction with 10a. When using PIDA at room temperature (Method A), there were no results. The use of Method D after reflux in ethanol for 2 days led to the isoxazole 13a in 55% yield (Table 2, entry 7). The same reaction under Method C afforded isoxazole 13a in 40% yield (Table 2, entry 8). The attempts to trigger a reaction of 10b with oxime 8 under all the methods examined (Methods A, B, C, and D) were unsuccessful, leaving the 4-propargylaminocoumarin (10b) unaffected. The results suggested that Methods B, C, and D produce similar moderate-to-good results for the 4-substituted propargyl coumarin derivatives.

Scheme 3. Reaction conditions for (i), Method B; (ii), Method C; and (iii) Method D are referred to in Scheme 1 and Scheme 2 captions.

Scheme 3. Reaction conditions for (i), Method B; (ii), Method C; and (iii) Method D are referred to in Scheme 1 and Scheme 2 captions.

2.1. Biology

Isoxazole derivatives of coumarin or pyridine have been proven to exhibit anticancer, antioxidant, and anti-inflammatory properties [27,28,29,40]. Therefore, we decided to evaluate the in vitro behavior of these new coumarin–isoxazole–pyridine hybrids as inhibitors of lipid peroxidation and as anti-inflammatories through the inhibition of soybean lipoxygenase (sLOX).

We investigated the antioxidant activity of the compounds as inhibitors of the lipid peroxidation of linoleic acid sodium induced by alkyl peroxy free radicals produced by 2,2-azobis(2-amidinopropane) hydrochloride (AAPH). The water-soluble AAPH generates in vitro peroxyl free radicals through spontaneous thermal decomposition. The derived experimental conditions resembled cellular lipid peroxidation due to the activity of the formed radicals. Trolox was used as a reference compound for comparative purposes (

Table 3. In vitro activities of compounds. Inhibition of soybean lipoxygenase (LOX). (%)/IC₅₀ µM. % Inhibition of lipid peroxidation (ILP) at 100 µM.

Entry	Compounds 1	Clog P 2	LOX(%)/ IC50 µM	ILP (%)
1	3a	2.27	no	66
2	3b	2.77	no	0.6
3	7a	2.27	10 µM	42
4	7b	2.27	no	2
5	9a	2.77	38	83.6
6	9b	2.77	no	86.6
7	11a	2.27	no	86
8	11b	1.95	no	66
9	12a	2.01	10	72
10	12b	1.95	18	90.4
11	13a	2.01	5 µM	44.6
12	NDGA		0.45 μΜ
13	Trolox			93

¹ Compounds tested at 100 µM. Values are means ± SD of three or four different. determinations. Means within each column differ significantly (p < 0.05); no result was given under the reported experimental conditions. ² Biobyte BioByte Corporation, C-QSAR database, 201 W Fourth Str., Suite # 204, Claremont. CA 91711-4707, USA.

). Among the hybrids, 12b, which is a 4-substituted amine, presented the highest activity (90.4%), whereas 9b, 11a, 9a, 12a, 11b, and 3a followed with 86.6, 86, 83, 72, and 66% anti-lipid peroxidation ability, respectively. Additionally, 13a and 7a exhibited lower activities, while 3b was inactive. Lipophilicity does not seem to influence activity. The inhibition of LOX was performed by the UV absorbance-based enzyme assay [68]. The IC₅₀ inhibition values in Table 3 showed two potent inhibitors, 13a and 7a. Both are ethers from the 7- or 4-position of the coumarin ring and are conjugated to a pyridyl group. The 4-pyridyl derivative 13a was highly active (IC₅₀ = 5 µM). No/low inhibition was shown by the other derivatives. The corresponding amino-substituted derivatives do not possess any activity. It seems that bulk and steric factors are important, and that the stereochemistry of the derivatives influences inhibition more than lipophilicity. Molecular volume and molar refractivity are two physicochemical parameters expressing the overall volume/size and stereochemistry of the molecules, and they play an important role in LOX inhibition. This agrees with previous results presented in a comparative QSAR study on LOX inhibitors [69]. The findings of the publication support that steric factors are important for LOX inhibition since there are steric requirements for the catalytic site of the enzyme.

2.2. Biochemistry

Cytotoxic activity against three different cancer cell lines was examined for the more potent isoxazole derivatives 7a, 9a, 12b, and 13a. HeLa from cervical cancer, HT-29 from colon cancer, and H1437 from lung cancer were utilized to assess the cytotoxic activity of those compounds by using the colorimetric method 3-(4,5-dimethylthiazol-2yl)-2,5 diphenyl tetrazolium bromide (MTT) [70]. The results were expressed as EC₅₀ (the concentration that causes 50% loss of cell viability) (

Table 4. Half maximal effective concentration (EC₅₀ Values) of 7a, 9a, 12b, and 13a in HeLa, HT-29, and H1437 cancer cell lines. Results are presented as a means ±SE of three independent experiments.

Entry	Compound	HeLa, EC50 (μM)	HT-29, EC50 (μM)	H1437, EC50 (μM)
1	7a	>100	>100	>100
2	9a	>100	>100	>100
3	12b	38.1 ± 2.1	96.5 ± 6.6	47.3 ± 3.1
4	13a	44.2 ± 1.9	65.8 ± 5.4	74.8 ± 4.4

). The results showed that 7a and 9a exhibited EC₅₀ values greater than 100 μM in all three cell lines, indicating low cytotoxicity (Table 4, entries 1,2). In contrast, 12b demonstrated the highest cytotoxicity in HeLa cells (EC₅₀ = 38.1 μM) and a similar effect in H1437 cells (EC₅₀ = 47.3 μM), whereas it was less effective in HT-29 cells (EC₅₀ = 96.5 μM) (Table 4, entry 3). Similarly, 13a showed moderate cytotoxicity, with EC₅₀ values of 44.2 μM, 65.8 μM, and 74.8 μM in HeLa, HT-29, and H1437 cells, respectively (Table 4, entry 4).

3. Materials and Methods

3.1. Materials

All the chemicals were purchased from either Sigma-Aldrich Chemie GmbH (Eschenstr. 5, 82024 Taufkirchen (bei Munchen), Germany) or Merck KGaA, (Frankfurter Strasse 250, 64293 Darmstadt, Germany). Melting points were determined with a Kofler hot stage apparatus and are uncorrected. IR spectra were obtained with a PerkinElmer Spectrum BX spectrophotometer as Nujol mulls. NMR spectra were recorded with an Agilent 500/54 (DD2) (500 MHz and 125 MHz for ¹H and ¹³C, respectively) using TMS as an internal standard. J values are reported in Hz. Mass spectra were determined with an LCMS-2010 EV instrument (Shimadzu, Kyoto, Japan) under electrospray ionization (ESI) conditions. HRMS (ESI-MS) were recorded with a ThermoFisher Scientific (168 Third Avenue, Waltham, MA 02451, USA) model LTQ Orbitrap Discovery MS. Silica gel No. 60 (Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany) was used for column chromatography.

3.2. Chemistry

General Procedure of the 1,3-dipolar Cycloaddition Reactions of Propargyl Coumarins with Pyridine Aldoximes and Synthesis of (3-(pyridin-2-yl)isoxazol-5-yl)methoxy)-2H-chromen-2-one (3a)

Method A: A total of 0.1 g (0.5 mmol) of 7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (1a) was dissolved in methanol (4 mL) under stirring at room temperature. 0.161 g (0.5 mmol) of PIDA was then added. Then, 56 mg (0.455) mmol of picolinaldehyde oxime (2) was dissolved in methanol (4 mL), and the solution was added dropwise to the solution of alkyne over a period of 2 h. The reaction was monitored by TLC (hexane:ethyl acetate (EA) (3:1)).

The reaction was completed 1 h after the addition of oxime. The crude mixture was evaporated, and the residue was separated by column chromatography (hexane:EA (3:1)to EA) to afford 88 mg (60%) of 3a and 22 mg (20%) of 4.

Method B: A total of 0.1 g (0.5 mmol) of 1a was dissolved in ethanol (4 mL) under stirring at room temperature. A total of 0.161 g (0.5 mmol) of PIDA and 56 mg (0.455 mmol) of 2 were added, and the reaction mixture was irradiated under MW irradiation at 120 °C for 1 h. The reaction was monitored by TLC (hexane:EA (3:1)). The mixture was filtered, and the precipitate was washed with hexane (3 × 3 mL) and dried to give 70 mg (48%) of 3a. The filtrate was evaporated and purified by column chromatography (hexane:EA (3:1))to EA to give 18 mg (16%) of 4 and 10 mg (9%) of 5.

Method C: In a solution of 0.1 g (0.5 mmol) of 1a in acetonitrile (8 mL), 56 mg (0.455 mmol) 2) and 0.006 mL (52 mg, 0.5 mmol) of TBN were added under N₂ atmosphere at room temperature. The reaction was monitored by TLC (hexane:EA (3:1)). The reaction was refluxed for 18 h. After the completion of the reaction, as indicated by TLC, water (10 mL) was added. The precipitate formed was purified by column chromatography (hexane:EA (1:1) to EA) to give 50 mg (34%) of 3a. The filtrate was extracted with EA (3 × 15 mL), and the combined organic layers were washed once with brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude mixture was purified by column chromatography (hexane:EA (3:1) to EA) to afford 33 mg (32%) of 5.

Method D: A total of 0.1 g (0.5 mmol) of 7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (1a) was dissolved in methanol (4 mL) under stirring at room temperature. A total of 0.161 g (0.5 mmol) of PIDA was then added, followed by 0.0168 mL (28.5 mg (0.25 mmol)) of TFA. A total of 56 mg (0.455) mmol of isonicotine aldehyde oxime (6) was dissolved in methanol (22 mL), and the solution was added dropwise to the solution of alkyne over a period of 2 h. The reaction was monitored by TLC (hexane:EA (3:1)). The reaction was refluxed for 2 days after the addition of oxime. The crude mixture was evaporated, and the residue was separated by column chromatography (hexane:EA (3:1) to EA) to afford 35 mg (24%) of 9a.

• (3-(Pyridin-2-yl)isoxazol-5-yl)methoxy)-2H-chromen-2-one (3a)

White solid, m.p. 178–179 °C, (hexane/ethyl acetate). ¹H-NMR (500 MHz, CDCl₃) δ: 5.30 (s, 2H), 6.29 (d, J = 9.5 Hz, 1H) 6.93 (s, 1H), 6.93 (d, J = 8.1 Hz, 1H), 7.04 (s, 1H), 7.33–7.38 (m, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 9.5 Hz, 1H), 7.81 (t, J = 7.2 Hz, 1H), 8.09 (d, J = 7.8 Hz, 1H), 8.68 (d, J = 4.2 Hz, 1H). ¹³C-NMR (126 MHz, CDCl₃) δ: 61.6, 102.2, 103.1, 112.9, 113.6, 114.1, 121.9, 124.9, 129.2, 137.1, 143.3, 148.1, 149.9, 155.8, 160.8, 161.0, 163.6, 167.4. IR (Nujol): 3086, 1728, 1629 cm⁻¹. LC-MS (ESI): (m/z): 343 [M + Na]⁺. HRMS (ESI): m/z calcd. for C₁₈H₁₂N₂O₄Na: 321.0870 (M + Na)⁺; found: 321.0898.

• 4-Methyl-7-((3-(pyridin-4-yl)isoxazol-5-yl)methoxy)-2H-chromen-2-one (3b).

Mass of 99 mg (65% under Method A), 65 mg (43% under Method B), 67 mg (44% under Method C), white solid, m.p. 173–175 °C, (hexane/ethyl acetate). ¹H NMR (300 MHz, CDCl₃) δ: 2.41 (s, 3H), 5.31 (s, 2H), 6.17 (s, 1H), 6.83–7.0 (m, 2H), 7.29 (s, 1H), 7.45– 7.53 (m, 1H), 7.55 (d, J = 8.7 Hz, 1H), 7.95 (t, J = 7.0 Hz, 1H), 8.20 (d, J = 7.9 Hz, 1H), 8.73 (d, J = 4.4 Hz, 1H). ¹³C-NMR (126 MHz, DMSO-d₆) δ: 18.2, 60.6, 101.8, 103.4, 11.7, 112.5, 113.9, 121.4, 125.3, 126.7, 137.6, 147.3, 150.0, 153.4, 154.6, 160.0, 160.3, 162.9, 168.1. IR (Nujol): 3085, 1725, 1621 cm⁻¹. LCMS (ESI): (m/z): 357 [M + Na]⁺. HRMS (ESI): m/z calcd. for C₁₉H₁₄N₂O₄H: 335.1026 (M + H)⁺; found: 335.1054.

7-((3-(Pyridin-3-yl)isoxazol-5-yl)methoxy)-2H-chromen-2-one (7a). Mass of 89 mg (61 % under Method C), white solid, m.p. 169–171 °C, (hexane/ethyl acetate). ¹H NMR (500 MHz, DMSO-d₆) δ: 5.51 (s, 2H), 6.34 (d, J = 9.5 Hz, 1H), 7.09 (dd, J = 8.6, 2.5 Hz, 1H), 7.20 (d, J = 2.4 Hz, 1H), 7.37 (s, 1H), 7.58 (dd, J = 8.0, 4.8 Hz, 1H), 7.69 (d, J = 8.7 Hz, 1H), 8.02 (d, J = 9.5 Hz, 1H), 8.30 (d, J = 7.9 Hz, 1H), 8.71 (dd, J = 4.8, 1.5 Hz, 1H), 9.10 (d, J = 1.6 Hz, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 60.9, 101.8, 102.8, 109.6, 112.9, 113.1, 124.3, 124.4, 129.7, 134.4, 144.3, 147.5, 151.2, 155.2, 159.9, 160.2, 160.5, 168.3. IR (Nujol): 3084, 1710, 1625 cm⁻¹. LCMS (ESI): (m/z): 321 [M + H]⁺, 343 [M + Na]⁺. HRMS (ESI): m/z calcd. for C₁₈H₁₂N₂O₄H: 321.0870 (M + H)⁺; found: 321.0866.

4-Methyl-7-((3-(pyridin-3-yl)isoxazol-5-yl)methoxy)-2H-chromen-2-one (7b). Mass of 81 mg (53 % under Method C), white solid, m.p. 150–152 °C, (hexane/ethyl acetate). ¹H NMR (500 MHz, DMSO-d₆) δ: 2.40 (s, 3H), 5.29 (s, 2H), 6.17 (s, 1H), 6.75 (s, 1H), 6.92 (d, J=2.0 Hz 1H), 6.95 (dd, J = 8.8, 2.0 Hz, 1H), 7.42 (dd, J = 7.4, 5.0 Hz, 1H), 7.55 (d, J = 8.7 Hz, 1H), 8.16 (d, J = 7.8 Hz, 1H), 8.70 (d, J = 3.3 Hz, 1H), 9.02 (s, 1H). ¹³C NMR (126 MHz, CDCl₃) δ: 18.8, 61.5, 101.8, 102.1, 112.5, 112.9, 114.8, 124.0, 124.9, 126.1, 134.3, 148.0, 151.3, 152.4, 155.2, 160.2, 160.5, 161.1, 168.1. IR (Nujol): 3090, 1710, 1620 cm⁻¹. LCMS (ESI): (m/z): 357 [M + Na]⁺. HRMS (ESI): m/z calcd. for C₁₉H₁₄N₂O₄H: 335.1026 (M + H)⁺; found: 335.1018.

7-((3-(Pyridin-4-yl)isoxazol-5-yl)methoxy)-2H-chromen-2-one (9a). Mass of 35 mg (24 % under Method D), 61 mg (42% under Method C), white solid, m.p. 181–183 °C, (hexane/ethyl acetate). ¹H NMR (500 MHz, DMSO-d₆) δ: 5.38 (s, 2H), 6.20 (d, J = 9.5 Hz, 1H), 6.95 (dd, J = 8.6, 2.4 Hz, 1H), 7.00 (d, J = 2.4 Hz, 1H), 7.18 (s, 1H), 7.50(dd, J = 8.6, 3.5 Hz, 1H), 7.78 (dd, J = 9.2, 4.6 Hz, 1H), 7.96 (d, J = 4.1 Hz, 2H), 8.77 (d, J = 4.1 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃/DMSO-d₆) δ: 60.6, 101.4, 102.4, 106.3, 112.3, 112.88, 112.94, 121.6, 129.0, 143.3, 147.8, 155.1, 159.5, 159.9, 160.1. IR (Nujol): 3090, 1715, 1628 cm⁻¹. LCMS (ESI): (m/z): 321[M + H]⁺. HRMS (ESI): m/z calcd. for C₁₈H₁₂N₂O₄H: 321.0870 (M + H)⁺; found: 321.0857. m/z calcd. for C₁₈H₁₂N₂O₄Na: 343.0689 (M + Na)⁺; found: 343.0684.

4-Methyl-7-((3-(pyridin-4-yl)isoxazol-5-yl)methoxy)-2H-chromen-2-one (9b). Mass of 68 mg (45% under Method C), white solid, m.p. 180–181 °C, (hexane/ethyl acetate). ¹H NMR (500 MHz, DMSO-d₆) δ: 2.41 (s, 3H), 5.53 (s, 2H), 6.25 (s, 1H), 7.10 (d, J = 8.7 Hz, 1H), 7.18 (s, 1H), 7.38 (s, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.87 (d, J = 5.1 Hz, 2H), 8.75 (d, J = 5.0 Hz, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 18.1, 60.8, 101.8, 102.9, 111.7, 112.5, 113.9, 120.9, 126.7, 128.2, 128.9, 135.4, 150.7, 153.3, 154.6, 160.0, 160.3, 160.5, 168.8. IR (Nujol): 3086, 1718, 1630 cm⁻¹. LCMS (ESI): (m/z): 357 [M+Na]⁺. HRMS (ESI): m/z calcd. for C₁₉H₁₄N₂O₄H: 335.1026 (M + H)⁺; found: 335.1015.

4-((3-(Pyridin-2-yl)isoxazol-5-yl)methoxy)-2H-chromen-2-one (11a). Mass of 64 mg (44% under Method A), 90 mg (62% under Method C), white solid, m.p. 164–165 °C, (hexane/ethyl acetate). ¹H NMR (500 MHz, DMSO-d₆) δ: 5.67 (s, 2H), 6.20 (s, 1H), 7.34 (s, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.54–7.59 (m, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.86 (d, J = 7.9 Hz, 1H), 7.99 (td, J = 7.7, 1.1 Hz, 1H), 8.07 (d, J = 7.8 Hz, 1H), 8.74 (d, J = 4.2 Hz, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 61.6, 91.7, 103.7, 114.8, 116.5, 121.5, 122.9, 124.4, 125.3, 133.0, 137.6, 147.2, 150.0, 152.8, 161.4, 163.0, 164.0, 166.9. IR (Nujol): 3070, 1725, 1620 cm⁻¹. LCMS (ESI): (m/z): 343 [M + Na]⁺. HRMS (ESI): m/z calcd. for C₁₈H₁₂N₂O₄H: 321.0870 (M + H)⁺; found: 321.0883.

4-(((3-(Pyridin-2-yl)isoxazol-5-yl)methyl)amino)-2H-chromen-2-one (11b). Mass of 80 mg (55% under Method B), whitish solid, m.p. 196–198 °C, (hexane/ethyl acetate). ¹H NMR (500 MHz, CDCl₃) δ: 5.38 (s, 2H), 5.83 (s, 1H), 7.13 (s, 1H), 7.28 (t, J = 7.6 Hz, 1H), 7.33 (d, J = 8.2 Hz, 1H), 7.38 (dd, J = 6.9, 5.3 Hz, 1H), 7.57 (d, J = 7.7 Hz, 1H), 7.72–7.90 (m, 1H), 8.11 (d, J = 7.8 Hz, 1H), 8.69 (d, J = 4.4 Hz, 1H). ¹³C NMR (126 MHz, CDCl₃) δ: 61.7, 91.6, 103.7, 115.3, 117.0, 121.9, 123.2, 124.3, 125.0, 133.0, 137.2, 147.9, 149.9, 153.5, 162.4, 163.7, 164.7, 165.7; IR (Nujol): 3288, 3085, 1711, 1629 cm⁻¹. LCMS (ESI): (m/z): 343 [M + H + Na]⁺. HRMS (ESI): m/z calcd. for C₁₈H₁₃N₃O₃H: 320,103 (M + H)⁺; found: 320.1057. m/z calcd. for C₁₈H₁₃N₃O₃Na: 342.0849 (M+Na)⁺; found: 342.0868.

4-((3-(Pyridin-3-yl)isoxazol-5-yl)methoxy)-2H-chromen-2-one (12a). Mass of 44 mg (30% under Method D), 48 mg (33% under Method C), white solid, m.p. 189–191 °C, (hexane/ethyl acetate). ¹H NMR (500 MHz, CDCl₃) δ: 5.38 (s, 2H), 5.84 (s, 1H), 6.85 (s, 1H), 7.31 (t, J = 7.5 Hz, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.46 (dd, J = 6.9, 4.8 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.86 (d, J = 7.9 Hz, 1H), 8.20 (d, J = 7.7 Hz, 1H), 8.73 (d, J = 3.3 Hz, 1H), 9.05 (s, 1H). ¹³C NMR (126 MHz, CDCl₃/ DMSO-d₆) δ: 61.2, 91.3, 102.7, 114.7, 116.0, 123.7, 127.7, 128.5, 132.3, 145.4, 148.6, 152.6, 157.2, 159.0, 161.3, 163.8, 166.5. IR (Nujol): 3080, 1712, 1630 cm⁻¹. LCMS (ESI): (m/z): 343 [M + Na]⁺. HRMS (ESI): m/z calcd. for C₁₈H₁₂N₂O₄H: 321.0870 (M + H)⁺; found: 321.0852.

4-(((3-(Pyridin-3-yl)isoxazol-5-yl)methyl)amino)-2H-chromen-2-one (12b). Mass of 9 mg (6% under Method D), 84 mg (56% under Method C), white solid, m.p. 197–199 °C, (hexane/ethyl acetate); ¹H NMR (500 MHz, DMSO-d₆) δ: 4.79 (s, 2H), 5.31 (s, 1H), 7.17 (s, 1 H), 7.34 (d, J = 8.4 Hz, 1H), 7.37 (t, J = 7.3 Hz, 1H), 7.53 (dd, J = 7.9, 4.8 Hz, 1H), 7.63 (t, J = 8.3 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 8.2–8.30 (m, 1H), 8.45 (s, 1H), 8.68 (d, J = 3.5 Hz, 1H), 9.07 (d, J = 1.3 Hz, 1H), ¹³C NMR (126 MHz, DMSO-d₆) δ: 38.1, 83.1, 100.9, 114.4, 117.0, 122.6, 123.6, 124.2, 124.5, 132.2, 134.1, 147.5, 151.2, 153.09, 153.12, 159.8, 161.4, 170.2; IR (Nujol): 3320, 3085, 1710, 1625. cm⁻¹. LCMS (ESI): (m/z): 342 [M + Na]⁺. HRMS (ESI): m/z calcd. for C₁₈H₁₃N₃O₃H: 320.1030 (M + H)⁺; found: 320.1030.

4-((3-(Pyridin-4-yl)isoxazol-5-yl)methoxy)-2H-chromen-2-one (13a). Mass of 80 mg (55% under Method D), 58 mg (40% under Method C), m.p. 167–169 °C, (hexane/ethyl acetate). ¹H NMR (500 MHz, DMSO-d₆) δ: 5.67 (s, 1H), 6.17 (s, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.44 (d, J = 8.3 Hz, 1H), 7.52 (s, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.83–7.92 (m, 1H), 8.77 (d, J = 4.8 Hz, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 61.9, 91.8, 103.1, 114.8, 116.5, 121.0, 123.0, 124.4, 133.0, 135.3, 150.7, 152.8, 160.7, 161.4, 163.9, 167.7. IR (Nujol): 3090, 1704, 1640 cm⁻¹. LCMS (ESI): (m/z): 321 [M + H]⁺. HRMS (ESI): m/z calcd. for C₁₈H₁₂N₂O₄H: 321.0870 (M + H)⁺; found: 321.0871.

3.3. Biological Experiments

The in vitro assays were performed at a concentration of 100 µM (a 10 mM stock solution in DMSO was used, from which several dilutions were made for the determination of IC₅₀ values) at least in triplicate, and the standard deviation of absorbance was less than 10% of the mean. The compounds were diluted in 0.1% DMSO under sonification in an appropriate buffer in several dilutions (Table 2). Statistical comparisons were made using the Student’s t-test. A statistically significant difference was defined as p < 0.05.

3.3.1. Inhibition of Linoleic Acid Peroxidation

The in vitro study was evaluated as reported previously by our group [40]. A total of 10 microliters of the 16 mM sodium linoleate solution were added to the UV cuvette containing 0.93 mL of a 0.05 M phosphate buffer, pH 7.4, pre-thermostated at 37 °C. The oxidation reaction was initiated at 37 °C under air by the addition of 50 μL of a 40 mM AAPH solution, which was used as a free radical initiator. Oxidation was carried out in the presence of the samples (10 μL from the stock solution of each compound) in the assay without antioxidants and monitored at 234 nm. Lipid oxidation was recorded in the presence of the same level of DMSO and served as a negative control. Trolox was used as the appropriate reference compound (Table 2).

3.3.2. Soybean Lipoxygenase Inhibition Study

The in vitro study was evaluated as reported previously by our group [59]. The tested compounds were incubated in a tris buffer pH 9, at room temperature, with sodium linoleate (0.1 mM) and 0.2 mL of enzyme solution (1/9 × 10⁻⁴ w/v in saline, 1000 U/mL) for 5 min, and after that, the inhibition was measured. The method was based on the conversion of sodium linoleate to 13-hydroperoxylinoleic acid at 234 nm by the appearance of the conjugated diene. Nor-dihydroguaeretic acid NDGA (IC₅₀ = 0.45 μM) was used as a reference compound. Different concentrations were used to determine the IC₅₀ values. A blank determination was used first to serve as a negative control. The results are given in Table 2.

3.4. Biochemical Experiments

3.4.1. Cell Culture

HeLa (cervical cancer), HT-29 (colorectal cancer), and H1437 (lang adenocarcinoma) cell lines were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle Medium (DMEM). All media were supplemented with 10% fetal bovine serum (FBS) and antibiotic/antimytotic as monolayers at 37 °C in a 5% CO₂ incubator in a humidified atmosphere.

3.4.2. Cytotoxicity Evaluation

Cell viability was assessed using the MTT assay. Briefly, cells were seeded in 96-well plates at a density of 3 × 10³ cells per well for HeLa and H1437 and 4 × 10³ cells per well for HT-29 in 100 μL of complete medium. After 24 h of incubation for cell attachment, the cells were treated with varying concentrations of test compounds (ranging from 10 to 100 μM) for 48 h. Following the treatment period, MTT colorimetric assay was performed as described before [62]. Cell viability was calculated as a percentage of untreated control. The half-maximal effective concentration (EC₅₀) values are defined as the concentration that causes a 50% reduction in cell viability relative to controls. All experiments were performed in triplicate and repeated independently at least three times. The ±SE values were calculated.

4. Conclusions

Coumarin–isoxazole hybrids connected to a pyridine framework have been synthesized in moderate-to-good yields by the 1,3-dipolar cycloaddition reaction of nitrile oxides, derived in situ from pyridine aldehyde oximes under oxidation by PIDA or TBN, with 4- or 7-propargyloxycoumarins or 4-propargylaminocoumarins. The 4-pyridyl hybrid 13a has been found to be a potent inhibitor of LOX with IC₅₀ = 5 µM, followed by the 3-pyridyl hybrid 7a with IC₅₀ = 10 µM. Since lipoxygenases and their catalysis products are related to carcinogenic processes such as cell proliferation, differentiation, and apoptosis, the potent hybrid 13a will be used as a lead compound for further theoretical structural modifications, and in vitro assays. Compounds 12b and 13a presented moderate to minimal impact on HeLa, HT-29, and H1437 cancer cells. However, hybrid 12b presented a combination of high antilipid peroxidation and a moderate impact on HeLa cells and is considered to lead to the design of new hybrids with a higher impact on HeLa. Further work is underway to delineate the role of these hybrids in inflammation.