Novel pleconaril derivatives: influence of substituents in the isoxazole and phenyl rings on the antiviral activity against enteroviruses

Abstract

Today, there are no medicines to treat enterovirus and rhinovirus infections. In the present study, a series of novel pleconaril derivatives with substitutions in the isoxazole and phenyl rings was synthesized and evaluated for their antiviral activity against a panel of pleconaril-sensitive and -resistant enteroviruses. Studies of the structure-activity relationship demonstrate the crucial role of the N,N-dimethylcarbamoyl group in the isoxazole ring for antiviral activity against pleconaril-resistant viruses. In addition, one or two substituents in the phenyl ring directly impact on the spectrum of antienteroviral activity. The 3-(3-methyl-4-(3-(3-N,N-dimethylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 10g was among the compounds exhibiting the strongest activity against pleconaril-resistant as well as pleconaril-susceptible enteroviruses with IC₅₀ values from 0.02 to 5.25 μM in this series. Compound 10g demonstrated markedly less CYP3A4 induction than pleconaril, was non-mutagenic, and was bioavailable after intragastric administration in mice. These results highlight compound 10g as a promising potential candidate as a broad spectrum enterovirus and rhinovirus inhibitor for further preclinical investigations.

Graphical Abstract:

1. Introduction

Enteroviruses and rhinoviruses belong to the Enterovirus genus of the Picornaviridae family. There are about 100 serotypes of enteroviruses species A-D and more than 160 serotypes of rhinoviruses species A-C [1]. Enteroviruses cause a wide range of acute and chronic diseases, such as foot-hand-and-mouth disease, myocarditis, acute flaccid myelitis, cardiomyopathy, poliomyelitis, encephalitis, meningitis, bronchiolitis and pneumonia [2, 3]. Rhinoviruses are the major cause of the common cold, a mild illness of the upper respiratory tract, but can also induce more severe lower respiratory tract illnesses, for example, bronchitis, sinusitis and pneumonia and can exacerbate asthma, cystic fibrosis and chronic obstructive pulmonary disease [4–8]. Vaccine development for these viruses is complicated by the multiplicity of serotypes. Today, there are only vaccines to prevent poliomyelitis caused by polioviruses [9], two inactivated enterovirus A71 vaccines used in China for prevention of hand-foot-and-mouth human disease [10] and vaccine against foot-and-mouth disease virus [11]. Thus, development of effective antiviral small molecules acting on the viral life cycle represents a real clinical unmet need.

Enteroviruses, rhinoviruses A and B [12], as well as rhinoviruses C [13] have a hydrophobic pocket in capsid protein 1. The hydrophobic pocket of the majority of enteroviruses and rhinoviruses A and B is filled with a fatty acid (so called “pocket factor”), whereas pocket of rhinoviruses C is filled with multiple bulky residues [13]. The pocket factor is released during viral attachment to host cells. During the attachment, conformational changes in the viral capsid trigger the release of viral genomic RNA. Inhibitors competing with the pocket factor for binding to the hydrophobic pocket (capsid-binding inhibitors) prevent virus attachment and/or uncoating. Among the most extensively studied drug candidates with this capsid-binding mechanism of action against enteroviruses are pleconaril, vapendavir and pocapavir. The synthesis, biological studies and causes of clinical trial failures of these capsid-binding inhibitors were described comprehensively in a recently published review [14].

Resistance of entero- and rhinoviruses toward capsid-binding inhibitors is most commonly linked to substitutions of amino acid residues forming the hydrophobic pocket [15]. In addition, different amino acid substitutions of isoleucine in position 207 in the viral capsid protein 1 (I1207K/R/M/T), a position not directly involved in the formation of the hydrophobic pocket of the pleconaril-sensitive coxsackievirus B3 97927 were shown to induce resistance under experimental conditions [16, 17]. Generally, known naturally occurring and treatment-triggered resistance should be taken into account during the development of effective antienteroviral agents.

Up to now, there have been no regulatory agency approved antienteroviral medicines for clinical uses. Current developments in this field have not met safety and quality requirements. For example, pleconaril was not approved by the FDA for the treatment of common cold due to set of adverse effects (headache, diarrhea and nausea), cytochrome P-450 (CYP3A4) induction (increasing ethinyl estradiol metabolism) and existence of pleconaril-resistant viruses [18]. In February 2017, it was reported that another clinical candidate vapendavir, failed a phase IIb due to insufficient efficacy against RVs as compared with placebo [19]. In addition, a very high rate of resistance was observed (44% in the pocapavir treated group vs 10% in the placebo treated group) in a randomized blinded placebo-controlled study where poliovirus-infected patients were treated with pocapavir [20]. Therefore, development of potent selective broad-spectrum antienteroviral agents remains one of the priorities of medicinal chemists in this field.

We have previously synthesized several new pleconaril and [(biphenyloxy)propyl]isoxazole analogues where pleconaril resistance of some entero- and rhinoviruses was overcome by unsubstituted analogues or by monosubstitution in the central phenyl ring [21, 22]. In a recently published review, we tracked the evolution of the synthetic development of some significant capsid-binding inhibitors [14]. The consistent study of the structure-activity relationship helped us to select a scaffold as the basis for compounds overcoming resistance and lowering the risk of CYP3A4 induction.

In the present work, we show the influence of substituents in the 3-position of pleconaril and isoxazole ring on inhibition activity against a set of selected pleconaril-sensitive and -resistant entero- and rhinoviruses. We continued to examine the impact of substituents in the phenyl ring of pleconaril derivatives on antienteroviral activity for a better understanding of structure-activity relationships to overcome viral resistance to capsid-inhibitors. In addition, we show that the most active compound synthesized in the present study is a much weaker inducer of the CYP3A4 enzyme than pleconaril. In addition, it did not exert mutagenicity and showed high bioavailability after intragastric administration.

2. Results and discussion

2.1. Chemistry and chemical characterization of new pleconaril derivatives

The syntheses of target compounds are presented in Schemes 1–6.

Scheme 1.

Synthesis of 3-(4-(3-(3-carbalkoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazoles 6a–i. Reagents and conditions: (a) 5-chloro-1-pentyne, K₂CO₃, KI, N-methylpyrrolidone, 65 °C, 24h; (b) NH₂OH·HCl, K₂CO₃, EtOH_abs, reflux, 12h; (c) (CF₃CO)₂O, pyridine, 80–90 °C, 2–3h; (d) Et₃N, dimethylformamide, 80–90 °C, 2–3h.

Scheme 6.

Synthesis of pyridinyl analogues. Reagents and conditions: (a) 4-pentyn-1-ol, K₂CO₃, N-methylpyrrolidone, 65 °C, 24h; (b) NH₂OH·HCl, K₂CO₃, EtOH_abs, reflux, 12h; (c) (CF₃CO)₂O, pyridine, 80–90 °C, 2–3h; (d) 5b, Et₃N, dimethylformamide, 80–90 °C, 2–3h; (e) dimethylamine solution 17 wt. % in dioxane, 50–60 °C, 12h

O-Alkylation of hydroxybenzonitriles 1a,c–i with 5-chloro-1-pentyne in presence of potassium carbonate and potassium iodide in N-methylpyrrolidone at 65 °C provided the corresponding 4-(pent-4-yn-1-yloxy)benzonitriles 2a,c–i in good yields (Scheme 1). Treatment of 2a,c–i with hydroxylamine hydrochloride and potassium carbonate in absolute ethanol gave amidoximes 3a,c–i. Interestingly, two intermediates, 3d (Me) and 3e (MeO), were present in the form of two tautomeric structures, which is observed by characteristic peaks in their ¹H NMR. It should also be noted that the presence or absence of tautometic structures in compounds 3a, c–i does not affect subsequent reactions. For cyclization into 1,2,4-oxadiazole compounds 3a,c–i were acylated with trifluoroacetic anhydride in pyridine with synthesis the appropriate (pent-4-yn-1-yloxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazoles 4a,c–i. Their reaction with 2-chloro-2-(hydroxyimino)acetic acid methyl ester 5a or 2-chloro-2-(hydroxyimino)acetic acid ethyl ester 5b in presence of triethylamine in dry dimethylformamide (DMF) led to 3-(4-(3-(3-carbalkoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazoles 6a–i.

3-(3,5-Dimethyl-4-(3-(3-carbisopropoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 7 was synthesized according to the two step procedure based on available chemicals shown in Scheme 2. Basic hydrolysis of previously prepared 3-(3,5-dimethyl-4-(3-(3-carbethoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 6b gave the corresponding acid and its subsequent Fischer esterification with i-propanol provided the target compound 7.

Scheme 2.

Synthesis of 3-(3,5-dimethyl-4-(3-(3-carbisopropoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 7. Reagents and conditions: (a) 1. LiOH, MeOH/H₂O, 2. iPrOH, H₂SO_4cat, reflux.

As shown in Scheme 3, unsubstituted or substituted (((phenylisoxazolyl)propoxy)phenyl)oxadiazoles 9a–c were prepared by cyclization of intermediate 4a with the corresponding benzaldehyde oximes 8a–c in the presence of N-chlorosuccinimide, triethylamine and pyridine as catalyst in dry dimethylformamide.

Scheme 3.

Synthesis of unsubstituted or substituted (((phenylisoxazolyl)propoxy)phenyl)oxadiazoles 9a–c. Reagents and conditions: (a) N-chlorosuccinimide, pyridine_cat, Et₃N, dimethylformamide, 80–90 °C.

Carbamoyl and substituted carbamoyl derivatives were prepared in two ways according to the Scheme 4. One way, (a), included treatment of 3-(3,5-dimethyl-4-(3-(3-carbethoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 6b with the corresponding amines in refluxing i-propanol to provide compounds 10a–b,m. Another way, (b), included substitution of the 3-carbethoxy group of (((isoxazolyl)propoxy)phenyl)oxadiazoles 6c–i with dimethylamine solution in water or dioxane to yield the respective dimethylcarbamoyl derivatives 10f–l. All of the reactions proceeded easily and quickly, and the final compounds 10a–m, which precipitate upon cooling of the reaction mixture as a well-formed solids were filtered and recrystallized from alcohols.

Scheme 4.

Synthesis of carbamoyl and substituted carbamoyl derivatives 10a–m. Reagents and conditions: (a) the corresponding amine, iPrOH, reflux, 18–24h; (b) dimethylamine solution 33 wt. % in H₂O or 17 wt. % in dioxane, EtOH, 40–50 °C, 1h.

Reduction of nitro derivative 6f with stannum chloride dehydrate in ethanol gave the amino derivative 11 in excellent yield (Scheme 5). Firstly, this derivative was functionalized by acetic anhydride, 2,5-dimethoxyfuran and dimethylformamide-dimethylacetal (DMF-DMA) to provide the corresponding final compounds 12a–c. Also, derivate 11 was treated with dimethylamine solution in dioxane to gave 3-(3-amino-4-(3-(3-N,N-dimethylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 13, which was functionalized in the same manner as 12a,b to obtain target compounds 14a,b.

Scheme 5.

Synthesis of amino derivatives 11 and 13 and functionalized amino analogues 12a–c and 14a,b. Reagents and conditions: (a) SnCl₂·2H₂O, EtOH, rt; (b) Ac₂O, 80 °C or 2,5-dimethoxyfuran, AcOH or DMF-DMA, EtOH, reflux; (c) dimethylamine solution 33 wt. % in H₂O, EtOH, 40–50 °C, 1h; (d) Ac₂O, 80 °C or 2,5-dimethoxyfuran, AcOH

It was discovered that the derivative 10i, containing a nitro group in the phenyl ring and a N,N-dimethylcarbamoyl group in the isoxazole, was active towards tested pleconaril-sensitive and - resistant viruses (see Table 2). Based on the similarity of pyridine to nitrobenzene, it seemed interesting to us to study how the exchange of the nitrogroup on insertion a nitrogen atom in the phenyl ring will affect the inhibition activity of compounds.

Table 2.

Cytotoxicity, anti-coxsackievirus B3 and anti-rhinovirus activity of synthesized phenyl-substituted (((carbethoxyisoxazolyl)propoxy)phenyl)oxadiazoles and (((N,N-dimethylcarbamoyl-isoxazolyl)propoxy)phenyl)oxadiazoles

No	CC50, μM	IC50 towards coxsackievirus B3, μM					IC50 towards Rhinovirus, μM
		97927				Nancy	A2	B5	B14
		I1207 I1092	I1207K I1092	I1207M I1092	I1207T I1092
6c	>100	0.746	n/a	6.54	42.89	n/a	n/a	n/a	0.34
6d	35.11	0.062	n/a	0.61	1.23	18.96	n/a	n/a	0.52
6e	>100	0.490	n/a	3.41	37.29	n/a	59.71	19.44	0.66
6f	>100	0.148	n/a	0.18	3.99	n/a	47.66	n/a	2.93
6g	60.19	0.217	n/a	1.35	24.90	n/a	n/a	n/a	0.50
6h	28.44	0.03	n/a	0.01	1.04	n/a	4.89	n/a	n/a
6i	>100	1.189	n/a	10.05	43.78	n/a	5.63	n/a	2.68
10f	27.98	0.458	27.49	0.02	8.11	10.81	4.61	1.74	0.04
10g	13.38	0.02	4.79	0.01	0.01	2.76	0.86	5.25	0.09
10h	68.00	0.030	51.71	0.60	1.66	37.76	1.97	3.28	0.04
10i	22.48	0.003	6.46	0.001	0.03	1.66	0.67	7.63	0.28
10j	20.16	0.025	n/a	0.06	1.44	18.48	3.77	4.04	0.05
10k	19.23	0.16	7.18	0.16	0.54	2.24	0.32	n/a	6.76
10l	25.55	0.365	n/a	0.88	0.84	14.47	0.32	n/a	0.20
10m	>100	0.02	n/a	0.06	1.04	14.11	0.96	n/a	n/a
11	>100	20.02	n/a	n/a	n/a	n/a	n/a	n/a	14.27
12a	>100	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a
12b	57.26	10.22	n/a	n/a	14.29	n/a	n/a	n/a	n/a
12c	>100	19.95	n/a	n/a	n/a	n/a	n/a	n/a	29.26
13	90.82	4.47	51.58	2.08	14.31	27.83	18.17	n/a	0.33
14a	>100	30.14	n/a	16.07	73.16	n/a	n/a	n/a	18.14
14b	21.24	0.82	n/a	1.06	3.55	n/a	3.21	n/a	7.34

For this purpose, the synthesis of pyridinyl analogues 19 and 20 started from chloronicotinonitrile 15, which was O-alkylated with 4-pentyn-1-ol in the presence of potassium carbonate in N-methylpyrrolidone to yield 6-(pent-4-yn-1-yloxy)nicotinonitrile 16 (Scheme 6). Consistent treatment of 16 with hydroxylamine hydrochloride and potassium carbonate in ethanol and trifluoroacetic anhydride in pyridine gave the corresponding (pyridinyl)oxadiazole 18. Cyclization of this intermediate with 2-chloro-2-(hydroxyimino)acetic acid ethyl ester 5b in dry dimethylformamide yielded 3-(4-(3-(3-carbethoxy-isoxazol-5-yl)propoxy)pyridin-3-yl)-5-trifluoromethyl-1,2,4-oxadiazole 19. Finally, the second target compound 20 was obtained by nucleophilic substitution with dimethylamine solution in dioxane.

2.2. Cytotoxicity and in vitro antiviral activity of synthesized compounds

Aiming to identify selective inhibitors against pleconaril-sensitive and pleconaril-resistant enteroviruses and rhinoviruses, we performed cytotoxic as well as antiviral assays in HeLa cells. The maximum tested concentration was 100 μM.

To exclude unspecific antiviral effects based on cytotoxicity, the effect of test compounds on HeLa cell viability was analyzed after 72 h of compound treatment as described previously [23]. Six wells with HeLa cells without compound served as cells control on each test plate. The test was stopped by adding a crystal violet/formalin/methanol solution [24]. After dye elution, cell viability of individual compound-treated wells was evaluated as the percentage of the mean value of optical density from the cell controls, which was set 100% cell viability. The 50% cytotoxic concentration (CC₅₀) was defined as the compound concentration reducing the viability of cell controls by 50%.

The set of pleconaril-sensitive and -resistant enteroviruses applied in the antiviral assays, included coxsackieviruses B3 Nancy, coxsackieviruses B3 97927 wild type and 3 mutants thereof and 3 rhinovirus serotypes A2, B5, and B14. Whereas coxsackievirus B3 Nancy and rhinovirus B5 are naturally pleconaril-resistant and possess resistance-conferring amino acids within the hydrophobic pocket, coxsackievirus B3 97927, rhinoviruses A2 and B14 are pleconaril-sensitive [15, 25]. In contrast, the pleconaril-resistant mutants of coxsackievirus B3 97927 with resistance-conferring amino acids outside, but near to the hydrophobic pocket were selected in the presence of pleconaril under experimental conditions [17]. All test viruses replicated well in HeLa cells. Replication of coxsackieviruses B3 and rhinoviruses caused the complete destruction of infected, untreated HeLa cells (called a cytopathic effect). Thus, inhibition of cytopathic effect (CPE) by test compounds (corresponding to increase of cell viability) could be used as parameter for evaluation of antiviral activity. Compounds were added immediately before virus infection of HeLa cells. At 48 h or 72 h after infection with coxsackieviruses B3 or rhinoviruses, respectively, the test was stopped by adding a crystal violet/formalin/methanol solution [16, 23]. At this time a nearly complete cytopathic effect was observed microscopically in six virus-infected, untreated HeLa cells whereas a confluent HeLa cell monolayer was apparent in six uninfected, untreated cells controls. The percentage of antiviral activity of the tests compounds were calculated according to Pauwels et al. [26] using the following equation: antiviral activity = [(mean optical density of 6 cell controls - mean optical density of 6 virus controls)/(optical density of test - mean optical density of 6 virus controls)] × 100 %. The 50% inhibitory concentration (IC₅₀) was determined as the compound concentration reducing the virus-induced CPE by 50% (50% viability of mock-treated, uninfected).

2.2.1. The effect of substituents in the 3-position of isoxazole ring on the antiviral activity

Firstly, we evaluated the influence of replacement of the methyl group in the isoxazole ring of pleconaril with various substituents, such as carbamoyl, substituted carbamoyl and carbalkoxy groups, phenyl and substituted phenyl rings, on the viral inhibition. Biological results are shown in Table 1.

Table 1.

Cytotoxicity, anti-coxsackievirus B3 and anti-rhinovirus activity of pleconaril and synthesized (dimethyl((isoxazolyl)propoxy)phenyl)oxadiazoles

No	CC50, μM	IC50 towards coxsackievirus B3, μM					IC50 towards rhinovirus, μM
		97927				Nancy	A2	B5	B14
		I1207 I1092	I1207K I1092	I1207M I1092	I1207T I1092
GH	n/t	592.34	664.07	681.76	725.20	422.68	n/t	n/t	n/t
P	26.25	0.01	n/a	2.05	3.97	n/a	0.04	n/a	0.07
6a	35.37	0.06	20.44	1.53	12.83	17.92	0.36	n/a	0.37
6b	17.21	0.07	11.12	0.94	20.40	n/a	n/t	n/t	n/t
7	17.93	0.19	n/a	4.09	n/a	n/t	2.43	n/t	0.43
9a	>100	0.14	n/a	0.28	1.69	n/t	0.76	n/a	0.53
9b	41.93	n/a	n/a	n/a	n/a	n/t	2.11	n/t	2.78
9c	>100	n/a	n/a	n/a	n/a	n/t	n/a	n/t	n/t
10a	>100	0.07	n/a	1.69	n/a	n/t	0.50	n/t	0.22
10b	>100	0.06	n/a	0.37	n/a	n/t	0.20	n/t	0.02
10c	>100	0.05	n/a	0.48	n/a	n/t	0.60	n/t	0.18
10d	>100	1.39	n/a	14.36	n/a	n/t	0.23	n/t	0.25
10e	13.20	0.02	6.33	0.03	1.01	n/t	0.12	n/t	0.12

CC₅₀ – 50% cytotoxic concentration, IC₅₀ – 50% inhibitory concentration, GH – guanidine hydrochloride, P – pleconaril, n/t – not tested, n/a – not active

Replacement of the methyl group of isoxazole ring with a carbalkoxy (6a, b, 7) and carbamoyl groups (10a–e) revealed compounds, which have activity comparable to the reference compound – pleconaril. The increasing length and volume of the side alkyl chain (Me → Et → iPr) of ester derivatives moderately reduced activity: a carbmethoxy (6a) inhibited pleconaril-susceptible and pleconaril-resistant viruses in IC₅₀ values from 0.06 to 20.44 μM, but at the same time no activity against pleconaril-resistant rhinovirus B5 was observed. It is interesting to note that compounds with carbamoyl and substituted carbamoyl groups (10a–e) were more active towards the coxsackievirus B3 97927 wild type and I1207M mutant than corresponding compounds with carbalkoxy groups (6a, b, and 7). In the case of coxsackievirus B3 I1207T inhibition confounding results of a structure-activity relationship were obtained: for 6a and 6b derivatives decreased activity was observed compared with pleconaril, and all of the carbamoyl derivatives, except for 10e, were totally inactive towards this mutant, whereas 10e was approximately 4-times more active than pleconaril (IC₅₀ = 1.01 μM for 10e and 3.97 μM for pleconaril). Compound 10d with a bulky substituent (benzylcarbamoyl) was significantly less active against the studied viruses compared with other carbamoyl derivatives and pleconaril. The small alkyl groups, such as methyl (10b) and ethyl (10c) in the carbamoyl derivative (10a) retained activity towards tested viruses (except for I1207K and I1207T, not active). Interestingly, the dimethylcarbamoyl derivative (10e) exhibited the most potent activity (coxsackievirus B3 Nancy and rhinovirus B5 not tested) in this series.

It is worth noting that derivatives with another bulky groups, such as phenyl (9a), dimethoxyphenyl (9b) and 4-(trifluoromethoxy)phenyl (9c) were completely inactive against the majority of viruses.

2.2.2. The effect of substituents in the phenyl ring on the antiviral activity

Secondly, we examined the influence of substituents with various properties, e.g. electronic, steric or hydrophilic/hydrophobic, in the central phenyl ring or replacement of the phenyl with a pyridine. Carbethoxy and dimethylcarbamoyl groups were selected in the capacity of the constant substituents in the isoxazole ring. Biological results were showed in Tables 2 and 3.

Table 3.

Cytotoxicity and antiviral activity of synthesized pyridinyl analogues

No	CC50, μM	IC50 towards coxsackievirus B3, μM					IC50 towards Rhinovirus, μM
		97927				Nancy	A2	B5	B14
		I1207 I1092	I1207K I1092	I1207M I1092	I1207T I1092
19	>100	10.26	n/a	n/a	n/a	n/a	n/a	n/a	1.99
20	48.84	5.95	22.01	5.93	17.38	22.17	5.83	4.28	0.12

It was shown that derivatives 6c-i exhibited similar activity against enteroviruses and reduced activity against rhinoviruses like pleconaril. However, 6f and 6h were more active towards two mutants coxsackievirus B3 (I1207M and I1207T) than pleconaril (IC₅₀ = 0.18 and 3.99 μM for 6f, 0.01 and 1.04 μM for 6h, 2.05 and 3.97 μM for pleconaril, respectively), whereas derivative 6d not only inhibited these viruses at a lower 50% concentration, but also was active against pleconaril-resistant coxsackievirus B3 Nancy (IC₅₀ = 18.96 μM).

We observed that derivatives with the dimethylcarbamoyl group in the isoxazole ring 10f–m, 13, 14a,b were more potent than with carbethoxy 6c–i, 11, 12a–c and the reference compound regardless of the substitution nature in the phenyl ring again. Whereas 6c with carbethoxy group in the 3-position of isoxazole and without any substituents in phenyl ring was inactive against pleconaril-resistant viruses (coxsackievirus B3 I1207K and Nancy, rhinovirus B5), the IC₅₀ of 10f with dimethylcarbamoyl group of isoxazole was 27.49, 10.81 and 1.74 μM for these viruses, respectively.

Introduction of second substituent in 5-position of phenyl ring (6i and 10l with 5-methoxy and 3-nitro groups in the phenyl ring) significantly reduced inhibition activity compared with monosubstituted derivatives 6f and 10i having 3-nitro group in the central ring.

It is interesting to note that the derivative 10m with the methylcarbamoyl group in isoxazole and the trifluoromethyl in phenyl ring had a CC₅₀ value more than 100 μM whereas the CC₅₀ of 10k with the dimethylcarbamoyl group in isoxazole and trifluoromethyl in the same position was 19.23 μM. Compound 10m exhibited no activity against coxsackievirus B3 I1207K mutant compared to 10k, which showed inhibition all of coxsackieviruses B3 with IC₅₀ values of 0.16–7.18 μM. In addition, 10m did not totally inhibit rhinoviruses B5 and B14, while 10k was not active against rhinovirus B5 only.

The functionalization of the 3-amino group showed that derivatives 12a–c and 14a,b having bulky substituents in the phenyl ring were weakly active or completely inactive towards Coxsackie virus serotypes. Surprisingly, derivatives 12a–c with carbethoxy group in isoxazole moderately inhibited rhinoviruses, including pleconaril-resistant rhinovirus B5, while 14a,b having the N,N-dimethylcarbamoyl group in the same position were weakly active against rhinoviruses A2 and B14 and inactive against rhinovirus B5.

The 2-methyl analogue 10g demonstrated the best activity towards pleconaril-resistant as well as pleconaril-susceptible viruses with IC₅₀ values of 0.02, 4.79, 0.01, 0.01, 2.76 (for coxsackieviruses B3 97927 wt, I1207K, I1207M, I1207T, Nancy, respectively), 0.86, 5.25 and 0.09 μM (for rhinoviruses A2, B5 and B14, respectively). The selectivity indexes (SI = CC₅₀ / IC₅₀) of the most promising compound in the series 10g are given in Table 4. In addition, the dose-response curves obtained in the eight biological assays with 10g are shown in Figure 2.

Table 4.

Cytotoxicity, anti-coxsackievirus B3, anti-rhinovirus activity and selectivity indexes of the most promising compound 10g

No	CC50, μM	IC50 towards coxsackievirus B3 (mean, μM) and SI										IC50 towards rhinovirus (mean, μM) and SI
		97927								Nancy		A2		B5		B14
		I1207/I1092		I1207K/I1092		I1207M/I1092		I1207T/I1092
		mean	SI	mean	SI	mean	SI	mean	SI	mean	SI	mean	SI	mean	SI	mean	SI
10g	13.38	0.02	669	4.79	2.8	0.01	1338	0.01	1338	2.76	4.9	0.86	15.6	5.25	2.6	0.09	149

Fig. 2.

Dose-dependent cytotoxicity (A), anti-coxsackievirus B3 (B), and anti-rhinovirus (C) activity of compound 10g in HeLa cells.

As for the pyridine analogues 19 and 20, it was clear that only the presence of one or another substituent in the isoxazole cycle played a critical role: thus, analogue 20 containing an N,N-dimethylcarbamoyl group showed moderate inhibition activity towards all of the studied viruses with IC₅₀ values from 0.12 to 22.17 μM, while analog 19 with the carbethoxy group was active only for coxsackievirus B3 wt with IC₅₀ = 10.26 μM and rhinovirus B14 with IC₅₀ = 1.99 μM.

(A) In the cytotoxicity assay compound 10g was added at the indicated concentrations to two-day-old confluent HeLa cell monolayers for 3 days. Thereafter, the cells were fixed and stained with a crystal-violet/formalin/methanol solution. After eluting the dye, the optical density of compound treated and untreated wells with HeLa cells was measured. The mean optical density of six mock-treated HeLa cell controls was set 100% cell viability when calculating the viability of compound 10g-treated cells. In (B) and (C) compound 10g was added at the indicated concentrations to HeLa cells immediately before infecting the cells with coxsackieviruses B3 (CVB3) or rhinoviruses (RV). The applied multiplicity of infection of CVB3 and RV resulted in a complete cytopathic effect of the six untreated HeLa cells (negative controls) after an incubation time of 48 h (CVB3) or 72 h (RV). Thereafter, the cells were fixed, stained, and destained as in the cytotoxicity assay and the optical density was measured. The mean optical density of six mock-treated, virus-infected cell controls was used as blank. The remaining mean optical density of six mock-treated, uninfected cell controls was set 100% cell viability when calculating the viability of compound 10g-treated, infected cells (antiviral activity). The mean percentage values of cell viability and standard deviation calculated from at least 3 independently performed cytotoxicity and antiviral assays is shown.

2.3. Pharmacokinetics of compound 10g after intragastric administration

We determined the pharmacokinetic properties of the most promising compound in the series 10g after intragastric administration of 100 mg/kg as 0.5 mL of prepared suspension in 0.5% CMC with one drop Tween-80 in mice. The 10g plasma concentration was determined using HPLC-MS. The pharmacokinetic curve of 10g is presented in Figure 3. The plasma concentration-time data were analyzed by applying the model-independent method with the ESTRIP computer program. Pharmacokinetic parameters are shown in Table 5 (for details see Experimental section). These results show that 10g has very high bioavailability. The concentration of 10g in blood and good value of the half-life favors this compound as potential drug candidate.

Fig 3.

Mean pharmacokinetic curve after single dose intragastric administration of 10g (n = 5; 100 mg/kg)

Table 5.

Pharmacokinetic parameters of 10g

AUC0-∞, ng/mL h	Cmax., ng/mL	Tmax., h	CL, h−1	kel h−1	T1/2, h	MRT, h	Vd, L
3015.5	1710.5±489.2	0.5	0.66	0.4373	1.58	2.15	1.52

2.4. Compound 10g demonstrated less CYP3A4 induction compared to pleconaril

As CYP3A4 induction is a key issue for pleconaril failure in the clinical trials, compound 10g was also tested in cryopreserved hepatocytes using rifampicin as positive control and midazolam as the substrate for the CYP3A4 enzyme. The induction experiment procedures were described in [27]. At 10 μM compound 10g induction of CYP3A4 was 1.4x (7% of control) vs pleconaril 2.7x (30% of control) and positive control 10 μM rifampicin 6.6x. At 1 μM compound 10g resulted in 1.2x (3% of control) vs pleconaril 1.7x (13% of control). These results demonstrate compound 10g is less likely to represent a CYP3A4 induction risk compared with pleconaril, which is a major differentiating factor that will impact clinical use.

2.5. Exclusion of genotoxicity of compound 10g

In the SOS-chromotest, none of the tested compounds appeared to be genotoxic in E. coli PQ37 at 10 mg/mL. Moreover, no inhibition of E. coli growth was detected (Table 6). The positive control, 4NQO, inhibited as expected the growth of E. coli PQ37, the zone of inhibition has 25 mm of diameter and the presence of a blue halo was detected indicative that X-Gal was metabolized (DNA damage). The negative control DMSO in this test, does not inhibit the growth of E. coli PQ37.

Table 6.

Mutagenicity of the compounds, DMSO and 4NQO. DMSO was used as negative control. 4NQO was used as positive control. All agents were tested at 10 mg/mL and DMSO 99%.

Compound	Concentration	Mutagenicity	Zone of inhibition, mm
10b	10 mg/mL	NO	0
10e	10 mg/mL	NO	0
10i	10 mg/mL	NO	0
10g	10 mg/mL	NO	0
10k	10 mg/mL	NO	0
4NQO	10 mg/mL	YES	25
DMSO	99%	NO	0

With regard to the potential mutagenicity of these compounds the results showed that none of them are mutagenic and they are not inhibiting the growth of Escherichia Coli bacteria.

3. Conclusion

Entero- and rhinoviruses cause a wide range of socially significant acute and chronic diseases. Up to now, there are three well-known capsid-binding agents against enteroviruses – pleconaril, vapendavir and pocapavir – but none of these inhibitors have been approved as antivirals because of CYP3A4 induction (pleconaril), lack of statistically significant antiviral effect (vapendavir) or high rate of viral resistance (pocapavir) observed in clinical trials. It is also worth noting that vaccine development for enteroviruses is complicated by the multiplicity of serotypes (about 260 serotypes). Thus, there is a real need to develop the potent broad-spectrum direct-acting antienteroviral small molecules.

For this purpose, a series of novel (((isoxazolyl)propoxy)phenyl)oxadiazoles and pyridinyl analogues was synthesized and evaluated for their antiviral activity against a panel of pleconaril-resistant and pleconaril-susceptible viruses – coxsackievirus B3 Nancy, coxsackievirus B3 97927 wild type and mutants thereof, rhinoviruses A2, B5 B14. The results showed that derivatives with N,N-dimethylcarbamoyl group in isoxazole ring were more active than the corresponding compounds with ethoxycarbonyl group in the same place. Significant impact of substituents in the central phenyl ring for antiviral activity was also found. Pleconaril resistance of coxsackievirus B3 97927 I1207K variant, coxsackievirus B3 Nancy and rhinovirus B5 was overcome by the unsubstituted analogue 10f, by monosubstitution with 3-methyl 10g, 3-nitro 10i and 3-F 10g or replacement of the phenyl with a pyridine 20. The 3-methyl derivative 10g inhibited all of the investigated viruses in micro- or nanomolar concentrations. Moreover, the lack of induction of CYP3A4 and the acceptable bioavailability after oral administration warrant further detailed preclinical investigations with 10g as well as assessment of other closely related viruses. We will next perform in vivo efficacy assessments in mice infected with representative viruses as well as performing further tests to confirm the mechanism of action is identical to pleconaril. The development of a drug for enteroviruses and rhinoviruses is an unmet medical need and we can learn from the failures of prior candidates in order to offer a higher probability of success in future clinical studies.

4. Experimental section

4.1. Chemistry

All reagents and solvents were purchased from commercial suppliers and used without further purification. ¹H spectra were measured on a Varian Unity +400 operated at 200–300 MHz. Chemical shifts were measured in DMSO-d₆ or CDCl₃, using tetramethylsilane as an internal standard. Mass spectra were obtained on a Finnigan SSQ-700 with direct injection. A Waters Micromass ZQ detector was used in ESI MS for identification of various products. Melting points were determined on Electrothermal 9001 and are uncorrected. Merck silica gel 60 F254 plates were used for analytical TLC; column chromatography was performed on Merck silica gel 60 (70–230 mesh).

Hydroxybenzonitriles 1a, c–e, g, h were synthesized from corresponding phenols by N-bromosuccinimide (NBS) bromination and subsequent change bromine atom to cyano group by copper (I) cyanide in dry dimethylformamide [28, 29]. 4-Hydroxy-3-methoxy-5-nitrobenzonitrile 1i was synthesized by nitration of 4-hydroxy-3-methoxybenzonitrile 1e [30]. 4-Hydroxy-3-nitrobenzonitrile 1f was obtained by nitration of 4-hydroxybenzonitrile with nitric acid/acetic acid [31].

2-Сhloro-2-(hydroxyimino)acetic acid methyl ester 5a and 2-chloro-2-(hydroxyimino)acetic acid ethyl ester 5b were synthesized from corresponding glycine ester hydrochloride by nitrosation with sodium nitrite and hydrochloric acid [32]. Substituted and unsubstituted benzaldehyde oximes 8a–c were obtained by oximation of corresponding benzaldehydes [33]. 6-Chloronicotinonitrile 17 was synthesized by dehydration of 6-chloronicotinamide with thionyl chloride [34, 35].

4.1.1. General procedure for the synthesis of compounds 2a,c–i

A mixture of corresponding hydroxybenzonitrile 1a,c–i (1 mol), finely divided K₂CO₃ (5 mol), KI (0.01 mol), 5-chloro-1-pentyne (1.5 mol) and N-methylpyrrolidone-2 was heated at 65 °C for 24 h. The cooled reaction mixture was treated by cold water and stirred for 3–4 h. The solid was collected and recrystallized from the corresponding solvent.

4.1.1.1. 3,5-Dimethyl-4-(pent-4-yn-1-yloxy)benzonitrile 2a

White solid, yield 74 %, mp 60–63 °C (EtOH). Mass (EI), m/z (I_relat.(%)): 213.2750 [M]⁺ (35). C₁₄H₁₅NO. ¹H NMR (DMSO-d₆): δ 1.91 (2H, dt, J = 6.5, 7.7, CH₂CH₂CH₂O), 2.23 (6H, s, (CH₃)₂Ph), 2.35 (2H, t, CH₂CH₂CH₂O), 2.85 (1H, s, CHCCH₂), 4.15 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.54–7.69 (2H, m, H3, H5) ppm.

4.1.1.2. 4-(Pent-4-yn-1-yloxy)benzonitrile 2c

White solid, yield 70 %, mp 86–89 °C (EtOH). Mass (EI), m/z (I_relat.(%)): 185.2218 [M]⁺ (44). C₁₂H₁₁NO. ¹H NMR (DMSO-d₆): δ 1.89 (2H, dt, J = 6.5, 7.7, CH₂CH₂CH₂O), 2.30 (2H, t, CH₂CH₂CH₂O), 2.81 (1H, s, CHCCH₂), 4.05 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.03 (2H, d, J = 8.8, H2, H6), 7.50 (2H, d, J = 8.8, H3, H5) ppm.

4.1.1.3. 3-Methyl-4-(pent-4-yn-1-yloxy)benzonitrile 2d

White solid, yield 92 %, mp 84–86 °C (EtOH). Mass (EI), m/z (I_relat.(%)): 199.2484 [M]⁺ (69). C₁₃H₁₃NO. ¹H NMR (DMSO-d₆): δ 1.91 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂), 2.16 (3H, s, СН₃Ph), 2.34 (2H, t, CH₂CH₂CH₂O), 2.81 (1H, s, CHCCH₂), 4.09 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.09 (1H, d, J = 8.3, H6), 7.55–7.65 (2H, m, H3, H5) ppm.

4.1.1.4. 3-Methoxy-4-(pent-4-yn-1-yloxy)benzonitrile 2e

White solid, yield 93 %, mp 103–106 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 215.2478 [M]⁺ (61). C₁₃H₁₃NO₂. ¹H NMR (DMSO-d₆): δ 1.92 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.36 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 3.81 (3H, s, CH₃O), 4.21 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.91 (1H, d, J = 8.1, H6), 7.47 (1H, d, J = 2.0, H3), 7.63 (1H, dd, J = 2.0, 8.1, H5) ppm.

4.1.1.5. 3-Nitro-4-(pent-4-yn-1-yloxy)benzonitrile 2f

White solid, yield 91 %, mp 118–121 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 230.2194 [M]⁺ (55). C₁₂H₁₀N₂O₃. ¹H NMR (DMSO-d₆): δ 1.91 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.32 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.79 (1H, s, CHCCH₂), 4.32 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.21 (1H, d, J = 8.3, H6), 8.15 (1H, dd, J = 1.5, 8.3, H5), 8.45 (1H, d, J = 1.5, H3) ppm.

4.1.1.6. 3-Fluoro-4-(pent-4-yn-1-yloxy)benzonitrile 2g

White solid, yield 98 %, mp 68–73 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 203.2123 [M]⁺ (55). C₁₂H₁₀FNO.¹H NMR (DMSO-d₆): δ 1.90 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.35 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 4.18 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.09 (1H, d, J = 8.3, H6), 7.75 (1H, dd, J = 1.5, 8.3, H5), 7.77 (1H, d, J = 1.5, H3) ppm.

4.1.1.7. 3-Trifluoromethyl-4-(pent-4-yn-1-yloxy)benzonitrile 2h

White solid, yield 100 %, mp 78–80 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 253.2198 [M]⁺ (56). C₁₃H₁₀F₃NO. ¹H NMR (DMSO-d₆): δ 1.91 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.34 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 4.21 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.10 (1H, d, J = 8.3, H6), 7.63 (1H, dd, J = 1.5, 8.3, H5), 7.72 (1H, d, J = 1.5, H3) ppm.

4.1.1.8. 3-Methoxy-5-nitro-4-(pent-4-yn-1-yloxy)benzonitrile 2i

White solid, yield 56 %, mp 105–107 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 260.2454 [M]⁺ (46). C₁₃H₁₂N₂O₄. ¹H NMR (DMSO-d₆): δ 1.93 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.36 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 3.77 (3H, s, CH₃O), 4.21 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.11 (1H, d, J = 8.3, H6), 7.64 (1H, dd, J = 1.5, 8.3, H5), 7.71 (1H, d, J = 1.5, H3) ppm.

4.1.2. General procedure for the synthesis of compounds 3a,c–i and 17

Compounds were obtained according to the previously described method [22].

4.1.2.1. N′-hydroxy-3,5-dimethyl-4-(pent-4-yn-1-yloxy)benzimidamide 3a

White solid, yield 81 %, mp 103–105 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 246.3049 [M]⁺ (36). C₁₄H₁₈N₂O₂. ¹H NMR (DMSO-d₆): δ 1.96 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O,), 2.29 (6H, s, (CH₃)₂Ph), 2.46 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 4.21 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.96 (1H, s, NOH), 5.08 (2H, brs, NH₂), 7.33 (2H, m, H3, H5) ppm.

4.1.2.2. N′-hydroxy-4-(pent-4-yn-1-yloxy)benzimidamide 3c

White solid, yield 79 %, mp 93–94 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 218.2518 [M]⁺ (45). C₁₂H₁₄N₂O₂. ¹H NMR (DMSO-d₆): δ 1.94 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.46 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 4.16 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.95 (1H, s, NOH), 5.06 (2H, brs, NH₂), 7.30 (2H, d, J = 8.8, H2, H6), 7.57 (2H, d, J = 8.8, H3, H5) ppm.

4.1.2.3. N′-hydroxy-3-methyl-4-(pent-4-yn-1-yloxy)benzimidamide 3d

White solid, yield 32 %, mp 110 °C (decomp.) (hexane/EtOAc). Mass (EI), m/z (I_relat.(%)): 232.2783 [M]⁺ (65). C₁₃H₁₆N₂O₂. ¹H NMR (CDCl₃): δ 2.05 (4H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.20 (3H, s, СН₃Ph), 2.46 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.86 (2H, s, CHCCH₂), 4.18 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.94 (1H, brs, NOH), 5.06 (1Н, brs, NHOH), 5.83 (1H, brs, NH), 6.81 (2H, m, NH₂), 7.25 (1H, s, NHOH), 7.39 (2H, d, J = 8.3, H6), 7.49 (2H, d, J = 8.3, H5), 7.67 (2H, s, H3) ppm.

4.1.2.4. N′-hydroxy-3-methoxy-4-(pent-4-yn-1-yloxy)benzimidamide 3e

White solid, yield 67 %, mp 82 °C (decomp.) (EtOH). Mass (EI), m/z (I_relat. (%)): 248.2777 [M]⁺ (66). C₁₃H₁₆N₂O₃. ¹H NMR (CDCl₃): δ 1.96 (4H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.46 (4H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (2H, s, CHCCH₂), 3.80 (3H, s, CH₃O), 4.20 (4H, t, J = 6.5, CH₂CH₂CH₂O), 4.98 (1H, brs, NOH), 5.08 (1Н, brs, NHOH), 5.85 (1H, brs, NH), 6.87 (2H, m, NH₂), 7.29 (1H, s, NHOH), 7.87 (2H, d, J = 8.3, H6), 7.29 (2H, dd, J = 1.5, 8.3, H5), 7.40 (2H, d, J = 1.5, H3) ppm.

4.1.2.5. N′-hydroxy-3-nitro-4-(pent-4-yn-1-yloxy)benzimidamide 3f

White solid, yield 71 %, mp 100–102 °C (hexane/EtOAc). Mass (EI), m/z (I_relat.(%)): 263.2493 [M]⁺ (61). C₁₂H₁₃N₃O₄. ¹H NMR (DMSO-d₆): δ 1.98 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.46 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 4.30 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.98 (1H, s, NOH), 5.09 (2H, brs, NH₂), 7.20 (1H, d, J = 8.3, H6), 7.84 (1H, dd, J = 1.5, 8.3, H5), 8.61 (1H, d, J = 1.5, H3) ppm.

4.1.2.6. N′-hydroxy-3-fluoro-4-(pent-4-yn-1-yloxy)benzimidamide 3g

White solid, yield 86 %, mp 93–97 °C (EtOH/H₂O). Mass (EI), m/z (I_relat. (%)): 236.2422 [M]⁺ (42). C₁₂H₁₃FN₂O₂. ¹H NMR (DMSO-d₆): δ 1.97 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.46 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 4.19 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.99 (1H, s, NOH), 5.05 (2H, brs, NH₂), 6.92 (1H, d, J = 8.3, H6), 7.45 (1H, dd, J = 1.5, 8.3, H5), 7.52 (1H, d, J = 1.5, H3) ppm.

4.1.2.7. N′-hydroxy-3-trifluoromethyl-4-(pent-4-yn-1-yloxy)benzimidamide 3h

White solid, yield 44 %, mp 118–121 °C (hexane/EtOAc). Mass (EI), m/z (I_relat.(%)): 286.2497 [M]⁺ (41). C₁₃H₁₃F₃N₂O₂. ¹H NMR (DMSO-d₆): δ 1.95 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.46 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 4.21 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.93 (1H, s, NOH), 5.07 (2H, brs, NH₂),7.10 (1H, d, J = 8.3, H6), 7.52 (1H, dd, J = 1.5, 8.3, H5), 7.61 (1H, d, J = 1.5, H3) ppm.

4.1.2.8. N′-hydroxy-3-methoxy-5-nitro-4-(pent-4-yn-1-yloxy)benzimidamide 3i

White solid, yield 67 %, mp 128–130 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 293.2753 [M]⁺ (37). C₁₃H₁₅N₃O₅. ¹H NMR (DMSO-d₆): δ 1.98 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.47 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 3.78 (3H, s, CH₃O), 4.29 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.95 (1H, s, NOH), 5.08 (2H, brs, NH₂), 7.03 (1H, d, J = 8.1, H2), 7.41 (1H, d, J = 2.0, H6) ppm.

4.1.2.9. N’-Hydroxy-6-(pent-4-yn-1-yloxy)nicotinimidamide 17

White solid, yield 38 %, mp 138–140 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 219.2398 [M]⁺ (48). C₁₁H₁₃N₃O₂. ¹H NMR (DMSO-d₆): δ 1.84 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.30 (2H, t, J = 7.6, CH₂CH₂CH₂O), 2.78 (1H, s, CHCCH₂), 4.29 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.98 (1H, s, NOH), 5.06 (2H, brs, NH₂), 6.76 (1H, d, J = 7.8, H3), 7.95 (1H, dd, J = 1.5, 7.8, H4), 8.45 (1H, d, J = 1.5, H6) ppm.

4.1.3. General procedure for the synthesis of compounds 4a,c–i and 18

Compounds were obtained according to the previously described method [22]. For compounds 4g and 18, the residue was purified by column chromatography with hexane: EtOAc = 2:1 as eluent. The product fractions were concentrated in vacuo. Compounds 4g and 18 were obtained as oils.

4.1.3.1. 3-(3,5-Dimethyl-4-(pent-4-yn-1-yloxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 4a

White solid, yield 61 %, mp 42–44 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 324.2977 [M]⁺ (49). C₁₆H₁₅F₃N₂O₂. ¹H NMR (DMSO-d₆): δ 1.98 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.19 (6H, s, (CH₃)₂Ph), 2.46 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 4.20 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.33 (2H, m, H3, H5) ppm.

4.1.3.2. 3-(4-(Pent-4-yn-1-yloxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 4c

White solid, yield 94 %, mp 43–45 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 296.2445 [M]⁺ (46). C₁₄H₁₁F₃N₂O₂. ¹H NMR (DMSO-d₆): δ 1.97 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.47 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 4.18 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.27 (2H, d, J = 8.8, H2, H6), 7.86 (2H, d, J = 8.8, H3, H5) ppm.

4.1.3.3. 3-(3-Methyl-4-(pent-4-yn-1-yloxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 4d

Colorless oil, yield 96 %. Mass (EI), m/z (I_relat. (%)): 310.2711 [M]⁺ (54). C₁₅H₁₃F₃N₂O₂. ¹H NMR (DMSO-d₆): δ 1.98 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.17 (3H, s, СН₃Ph), 2.46 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 4.19 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.08 (1H, d, J = 8.3, H6), 7.62–7.69 (2H, m, H3, H5) ppm.

4.1.3.4. 3-(3-Methoxy-4-(pent-4-yn-1-yloxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 4e

White solid, yield 78 %, mp 67–71 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 326.2705 [M]⁺ (64). C₁₅H₁₃F₃N₂O₃. ¹H NMR (DMSO-d₆): δ 1.98 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.44 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 3.84 (3H, s, CH₃O), 4.19 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.03 (1H, d, J = 8.3, H6), 7.41 (1H, dd, J = 1.5, 8.3, H5), 7.52 (1H, d, J = 1.5, H3) ppm.

4.1.3.5. 3-(3-Nitro-4-(pent-4-yn-1-yloxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 4f

White solid, yield 83 %, mp 82–84 °C (hexane/EtOAc). Mass (EI), m/z (I_relat. (%)): 341.2421 [M]⁺ (69). C₁₄H₁₀F₃N₃O₄. ¹H NMR (DMSO-d₆): δ 1.99 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.35 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.81 (1H, s, CHCCH₂), 4.26 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.51 (1H, d, J = 8.3, H6), 8.28 (1H, dd, J = 1.5, 8.3, H5), 8.50 (1H, d, J = 1.5, H3) ppm.

4.1.3.6. 3-(3-Fluoro-4-(pent-4-yn-1-yloxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 4g

Colorless oil, yield 91 %. Mass (EI), m/z (I_relat. (%)): 314.2350 [M]⁺ (72). C₁₄H₁₀F₄N₂O₂. ¹H NMR (DMSO-d₆): δ 1.97 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.47 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 4.22 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.17 (1H, d, J = 8.3, H6), 7.61 (1H, dd, J = 1.5, 8.3, H5), 7.74 (1H, d, J = 1.5, H3) ppm.

4.1.3.7. 3-(3-Trifluoromethyl-4-(pent-4-yn-1-yloxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 4h

White solid, yield 79 %, mp 37–40 °C (EtOH/H₂O). Mass (EI), m/z (I_relat. (%)): 364.2425 [M]⁺ (42). C₁₅H₁₀F₆N₂O₂. ¹H NMR (DMSO-d₆): δ 1.98 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.44 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 4.23 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.28 (1H, d, J = 8.3, H6), 7.70 (1H, dd, J = 1.5, 8.3, H5), 7.89 (1H, d, J = 1.5, H3) ppm.

4.1.3.8. 3-(3-Methoxy-5-nitro-4-(pent-4-yn-1-yloxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 4i

White solid, yield 83 %, mp 70–75 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 371.2681 [M]⁺ (47). C₁₅H₁₂F₃N₃O₅. ¹H NMR (DMSO-d₆): δ 1.98 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.44 (2H, t, J = 7.5, CH₂CH₂CH₂O), 2.77 (1H, s, CHCCH₂), 3.84 (3H, s, CH₃O), 4.19 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.03 (1H, d, J = 8.1, H3), 7.41 (1H, d, J = 2.0, H5) ppm.

4.1.3.9. 3-(6-(Pent-4-yn-1-yloxy)pyridin-3-yl)-5-trifluoromethyl-1,2,4-oxadiazole 18

Colorless oil, yield 66%. Mass (EI), m/z (I_relat. (%)): 297.2326 [M]⁺ (51). C₁₃H₁₀F₃N₃O₂. ¹H NMR (DMSO-d₆): δ 1.93 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂), 2.33 (2H, t, J = 7.6, CH₂CH₂CH₂O), 2.80 (1H, s, CHCCH₂), 4.42 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.03 (1H, d, J = 7.8, H3), 8.28 (1H, dd, J = 1.5, 7.8, H4), 8.82 (1H, d, J = 1.5, H6) ppm.

4.1.4. General procedure for the synthesis of compounds 6a–i and 19

To a solution of 5a or 5b (3 mmol) in DMF was added a solution of corresponding pentynyloxyphenyl-oxadiazoles 4a,c–i in DMF for 20–30 min and stirred for 40–50 min at room temperature. To the reaction solution was added Et₃N (3 mmol) in DMF for 2h at 80–90 °C. The mixture was stirred for 1–2h at 80–90 °C and for 12h at room temperature. The reaction mixture was diluted with water and extracted with ethyl acetate (3-times). The combined organic phases were washed with water, dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was treated by water and stored in the refrigerator for 2–4 h. Crystals were collected and recrystallized from the corresponding solvent.

4.1.4.1. 3-(3,5-Dimethyl-4-(3-(3-carbmethoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 6a

White solid, yield 46 %, mp 76–78 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 425.3585 [M]⁺ (45). C₁₉H₁₈F₃N₃O₅. ¹H NMR (DMSO-d₆): δ 2.18 (2H, dt, J = 6.5, 7.7, CH₂CH₂CH₂O), 2.30 (6H, s, (CH₃)₂Ph), 3.11 (2H, t, J = 7.7, CH₂CH₂CH₂O), 3.84 (2H, t, J = 6.5, CH₂CH₂CH₂O), 3.89 (3H, s, CH₃O), 6.80 (1H, s, isoxazole), 7.75 (2H, s, H3, H5) ppm.

4.1.4.2. 3-(3,5-Dimethyl-4-(3-(3-carbethoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 6b

White solid, yield 45 %, mp 74–76 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 439.3851 [M]⁺ (59). C₂₀H₂₀F₃N₃O₅. ¹H NMR (DMSO-d₆): δ 1.31 (3H, t, J = 7.1, CH₃CH₂O), 2.18 (2H, dt, J = 6.5, 7.7, CH₂CH₂CH₂O), 2.30 (6H, s, (CH₃)₂Ph), 3.11 (2H, t, J = 7.7, CH₂CH₂CH₂O), 3.86 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.35 (2H, q, J = 7.1, CH₃CH₂O), 6.81 (1H, s, isoxazole), 7.75 (2H, s, H3, H5) ppm.

4.1.4.3. 3-(4-(3-(3-Carbethoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 6c

White solid, yield 58 %, mp 71–75 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 411.3319 [M]⁺ (53). C₁₈H₁₆F₃N₃O₅. ¹H NMR (DMSO-d₆): δ 1.31 (3H, t, J = 7.1, CH₃CH₂O), 2.16 (2H, dt, J = 6.5, 7.5 CH₂CH₂CH₂O), 3.00 (2H, t, J = 7.5, CH₂CH₂CH₂O), 4.14 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.35 (2H, q, J = 7.1, CH₃CH₂O), 6.76 (1H, s, isoxazole), 7.15 (2H, d, J = 8.8, H2, H6), 8.00 (2H, d, J = 8.8, H3, H5) ppm.

4.1.4.4. 3-(3-Methyl-4-(3-(3-carbethoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 6d

White solid, yield 91 %, mp 66–68 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 425.3585 [M]⁺ (47). C₁₉H₁₈F₃N₃O₅. ¹H NMR (DMSO-d₆): δ 1.27 (3H, t, J = 7.1, CH₃CH₂O), 2.15–2.25 (5H, m, CH₂CH₂CH₂O, CH₃Ph), 3.02 (2H, t, J = 7.5, CH₂CH₂CH₂O), 4.18 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.35 (2H, q, J = 7.1, CH₃CH₂O), 6.78 (1H, s, isoxazole), 7.18 (1H, d, J = 8.3, H6), 7.80–7.90 (2H, m, H3, H5) ppm.

4.1.4.5. 3-(3-Methoxy-4-(3-(3-carbethoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 6e

White solid, yield 64 %, mp 75–77 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 441.3579 [M]⁺ (45). C₁₉H₁₈F₃N₃O₆. ¹H NMR (DMSO-d₆): δ 1.31 (3H, t, J = 7.1, CH₃CH₂O), 2.18 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 3.02 (2H, t, J = 7.5, CH₂CH₂CH₂O), 3.86 (3H, s, CH₃O), 4.13 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.35 (2H, q, J = 7.1, CH₃CH₂O), 6.78 (1H, s, isoxazole), 7.18 (1H, d, J = 8.1, H3), 7.51 (1H, d, J = 2.0, H6), 7.65 (1H, dd, J = 2.0, 8.1, H4) ppm.

4.1.4.6. 3-(3-Nitro-4-(3-(3-carbethoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 6f

White solid, yield 60 %, mp 98–100 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 456.0893 [M]⁺ (41). C₁₈H₁₅F₃N₄O₇. ¹H NMR (DMSO-d₆): δ 1.31 (3H, t, J = 7.1, CH₃CH₂O), 2.25 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 3.02 (2H, t, J = 7.5, CH₂CH₂CH₂O), 4.35 (2H, q, J = 7.1, CH₃CH₂O), 4.42 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.75 (1H, s, isoxazole), 7.52 (1H, d, J = 8.3, H6), 8.26 (1H, dd, J = 1.5, 8.3, H5), 8.50 (1H, d, J = 1.5, H3) ppm.

4.1.4.7. 3-(3-Fluoro-4-(3-(3-carbethoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 6g

White solid, yield 57 %, mp 68–75 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 429.3224 [M]⁺ (49). C₁₈H₁₅F₄N₃O₅. ¹H NMR (DMSO-d₆): δ 1.27 (3H, t, J = 7.1, CH₃CH₂O), 2.20 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 3.01 (2H, t, J = 7.5, CH₂CH₂CH₂O), 4.24 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.36 (2H, q, J = 7.1, CH₃CH₂O), 6.78 (1H, s, isoxazole), 7.45 (1H, t, J = 8.6, H3), 7.79–7.91 (2H, m, H4, H6) ppm.

4.1.4.8. 3-(3-Trifluoromethyl-4-(3-(3-carbethoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 6h

White solid, yield 99 %, mp 76–78 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 479.3299 [M]⁺ (46). C₁₉H₁₅F₆N₃O₅. ¹H NMR (DMSO-d₆): δ 1.31 (3H, t, J = 7.1, CH₃CH₂O), 2.17 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 3.01 (2H, t, J = 7.6, CH₂CH₂CH₂O), 4.25–4.40 (4H, m, CH₂CH₂CH₂O, CH₃CH₂O), 6.75 (1H, s, isoxazole), 7.51 (1H, d, J = 8.0, H6), 8.20 (1H, s, H3), 8.32 (1H, d, J = 8.0, H5) ppm.

4.1.4.9. 3-(3-Methoxy-5-nitro-4-(3-(3-carbethoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 6i

White solid, yield 100 %, mp 48–50 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 486.3555 [M]⁺ (53). C₁₉H₁₇F₃N₄O₈. ¹H NMR (DMSO-d₆): δ 1.32 (3H, t, J = 7.1, CH₃CH₂O), 2.13 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 3.01 (2H, t, J = 7.5, CH₂CH₂CH₂O), 4.01 (3H, s, CH₃O), 4.24 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.36 (2H, q, J = 7.1, CH₃CH₂O), 6.74 (1H, s, isoxazole), 7.86 (1H, d, J = 2.0, H3), 8.08 (1H, d, J = 2.0, H5) ppm.

4.1.4.10. 3-(4-(3-(3-Carbethoxy-isoxazol-5-yl)propoxy)pyridin-3-yl)-5-trifluoromethyl-1,2,4-oxadiazole 19

White solid, yield 10 %, mp 45–48 °C (hexane). Mass (EI), m/z (I_relat. (%)): 412.3200 [M]⁺ (62). C₁₇H₁₅F₃N₄O₅. ¹H NMR (DMSO-d₆): δ 1.28 (3H, t, J = 7.1, CH₃CH₂O), 2.18 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 3.00 (2H, t, J = 7.6, CH₂CH₂CH₂O), 4.28 (2H, q, J = 7.1, CH₃CH₂O), 4.45 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.75 (1H, s, isoxazole), 7.01 (1H, d, J = 7.8, H3), 8.29 (1H, dd, J = 1.5, 7.8, H4), 8.80 (d, 1H, J = 1.5, H6) ppm.

4.1.5. Synthesis of 3-(3,5-dimethyl-4-(3-(3-carbisopropoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 7

1. A solution of 6b (0.1 mmol) and LiOH (0.16 mmol) in ethanol-water mixture (1:1) was stirred for 3 h at room temperature. Reaction mixture was concentrated in vacuo. The remaining aqueous solution was acidified by the addition of 6 M hydrochloric acid. The precipitated white solid was collected and washed with water. The acid obtained was used without other purification.

2. A solution of acid obtained previously, one-two drops of H₂SO₄ and isopropanol was refluxed for 4 h. After completion of reaction (monitored by TLC) the reaction mixture was concentrated in vacuo. The precipitate was collected, washed with water and recrystallized from ethanol. White solid, yield 78 %, mp 69–74 °C (EtOH). Mass (EI), m/z (I_relat. (%)): 453.4117 [M]⁺ (54). C₂₁H₂₂F₃N₃O₅. ¹H NMR (DMSO-d₆): δ 1.42 (6H, d, J = 7.0, (CH₃)₂CH), 2.18 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.30 (6H, s, (CH₃)₂Ph), 3.11 (2H, t, J = 7.7, CH₂CH₂CH₂O), 3.84 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.87 (1H, sept, J = 7.0, (CH₃)₂CH), 6.80 (1H, s, isoxazole), 7.75 (2H, s, H3, H5) ppm.

4.1.6. General procedure for the synthesis of compounds 9a–c

To a solution of NCS (2 mmol) and 1–2 drops of pyridine in DMF was added a solution of the corresponding benzaldehyde oximes 8a–c (1 mmol) in DMF for 20 min and stirred for 1h at room temperature, then to a solution was added a solution 4a in DMF for 20 min. To resulted mixture was added Et₃N (2 mmol) in DMF for 1h at 80–90 °C and stirred for 3–4h at 80–90 °C. The reaction mixture was diluted with water and extracted with ethyl acetate (3-times). The combined organic phases were washed with water, dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was recrystallized from the corresponding solvent.

4.1.6.1. 3-(3,5-Dimethyl-4-(3-(3-phenylisoxazol-5-yl)propoxy)phenyl)-5-(trifluoromethyl)-1,2,4- oxadiazole 9a

White solid, yield 24 %, mp 74–76 °C (hexane). Mass (EI), m/z (I_relat.(%)): 443.4184 [M]⁺ (54). C₂₃H₂₀F₃N₃O₃. ¹H NMR (DMSO-d₆): δ 2.20 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.30 (6H, s, (CH₃)₂Ph), 3.10 (2H, t, J = 7.5, CH₂CH₂CH₂O), 3.92 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.93 (1H, s, isoxazole), 7.50 (2H, m, m-Ph, p-Ph), 7.75 (2H, s, H3, H5), 7.90 (2H, m, o-Ph) ppm.

4.1.6.2. 3-(4-(3-(3-(3,4-Dimethoxyphenyl)isoxazol-5-yl)propoxy)-3,5-dimethylphenyl)-5- (trifluoromethyl)-1,2,4-oxadiazole 9b

White solid, yield 38 %, mp 112–114 °C (EtOH). Mass (EI), m/z (I_relat.(%)): 503.4704 [M]⁺ (67). C₂₅H₂₄F₃N₃O₅. ¹H NMR (DMSO-d₆): δ 2.23 (2H, dt, J = 6.5, 7.5, CH₂CH₂CH₂O), 2.30 (6H, s, (CH₃)₂Ph), 3.14 (2H, t, J = 7.5, CH₂CH₂CH₂O), 3.80 (3H, s, CH₃O), 3.85 (3H, s, CH₃O), 3.92 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.83 (1H, s, isoxazole), 7.08 (1H, d, J = 8.0, H2’), 7.41 (1H, d, J = 1.5, H6’), 7.44 (1H, dd, J = 1.5, 8.0, H5’), 7.75 (2H, s, H3, H5) ppm.

4.1.6.3. 3-(3,5-Dimethyl-4-(3-(3-(4-(trifluoromethoxy)phenyl)isoxazol-5-yl)propoxy)phenyl)-5- (trifluoromethyl)-1,2,4-oxadiazole 9c

White solid, yield 39 %, mp 65–67 °C (EtOH/H₂O). Mass (EI), m/z (I_relat.(%)): 527.4158 [M]⁺ (49). C₂₄H₁₉F₆N₃O₄. ¹H NMR (DMSO-d₆): δ 2.22 (2H, dt, J = 6.5, 7.7, CH₂CH₂CH₂O), 2.30 (6H, s, (CH₃)₂Ph), 3.10 (2H, t, J = 7.7, CH₂CH₂CH₂O), 3.94 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.93 (1H, s, isoxazole), 7.50 (2H, d, J = 8.4, H2’, H6’), 7.75 (2H, s, H3, H5), 8.00 (2H, d, J = 8.4, H3’, H5’) ppm.

4.1.7. General procedure for the synthesis of compounds 10a–e,m

A mixture of 6b (1 mmol), the corresponding amine (2 mmol) and iPrOH was refluxed for 18–36h. After completion of reaction (monitored by TLC) the reaction mixture was cooled. The precipitate was collected by filtration and recrystallized from the corresponding solvent.

4.1.7.1. 3-(3,5-Dimethyl-4-(3-(3-carbamoyl-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4- oxadiazole 10a

White solid, yield 64%, mp 147–152 °С (EtOH). Mass (EI), m/z (I_relat.(%)): 410.3473 [M]⁺ (67). C₁₈H₁₇F₃N₄O₄. ¹H NMR (DMSO-d₆): δ 2.23 (2H, dt, J = 6.5, 7.7, CH₂CH₂CH₂), 2.30 (6H, s, (CH₃)₂Ph), 3.12 (2H, t, J = 7.7, CH₂CH₂CH₂O), 3.80 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.62 (1H, s, isoxazole), 7.75 (3H, m, H3, H5, NH), 8.07 (1H, brs, NH) ppm.

4.1.7.2. 3-(3,5-Dimethyl-4-(3-(3-methylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl- 1,2,4-oxadiazole 10b

White solid, yield 67 %, mp 121–123 °С (EtOH). Mass (EI), m/z (I_relat.(%)): 424.3739 [M]⁺ (78). C₁₉H₁₉F₃N₄O₄. ¹H NMR (DMSO-d₆): δ 2.13 (2H, dt, J = 6.5, 7.7, CH₂CH₂CH₂O), 2.30 (6H, s, (CH₃)₂Ph), 2.75 (3H, d, J = 4.0, NCH₃), 3.12 (2H, t, J = 7.7, CH₂CH₂CH₂O), 3.82 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.70 (1H, s, isoxazole), 7.75 (2H, s, H3, H5), 8.60 (1H, br.d, J = 4.0, NH) ppm.

4.1.7.3. 3-(3,5-Dimethyl-4-(3-(3-ethylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl- 1,2,4-oxadiazole 10c

White solid, yield 73%, mp 110–112 °С (EtOH). Mass (EI), m/z (I_relat.(%)): 438.4007 [M]⁺ (59). C₂₀H₂₁F₃N₄O₄. ¹H NMR (DMSO-d₆): δ 1.22 (3H, t, J = 7.1, CH₃CH₂), 2.13 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.30 (6H, s, (CH₃)₂Ph), 3.10 (2H, q, J = 7.1, CH₃CH₂N), 3.14 (2H, t, J = 7.6, CH₂CH₂CH₂O), 3.92 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.83 (1H, s, isoxazole), 7.70 (2H, s, H3, H5) ppm.

4.1.7.4. 3-(3,5-Dimethyl-4-(3-(3-benzylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl- 1,2,4-oxadiazole 10d

White solid, yield 52 %, mp 102–104 °С (EtOH). Mass (EI), m/z (I_relat.(%)): 500.4699 [M]⁺ (42). C₂₅H₂₃F₃N₄O₄. ¹H NMR (DMSO-d₆): δ 2.13 (2H, dt, J = 6.5, 7.7, CH₂CH₂CH₂O), 2.30 (6H, s, (CH₃)₂Ph), 3.14 (2H, t, J = 7.7, CH₂CH₂CH₂O), 3.92 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.53 (2H, s, NCH₂), 6.85 (1H, s, isoxazole), 7.26–7.31 (5H, m, Ph), 7.70 (2H, s, H3, H5) ppm

4.1.7.5. 3-(3,5-Dimethyl-4-(3-(3-N,N-dimethylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5- trifluoromethyl-1,2,4-oxadiazole 10e

White solid, yield 33 %, mp 77–85 °С (EtOH). Mass (EI), m/z (I_relat.(%)): 438.4005 [M]⁺ (81). C₂₀H₂₁F₃N₄O₄. ¹H NMR (DMSO-d₆): δ 2.20 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.30 (3H, s, CH₃Ph), 2.50 (3H, s, CH₃Ph), 2.88–3.14 (8H, m, N(CH₃)₂, CH₂CH₂CH₂O), 3.87 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.54 (1H, s, isoxazole), 7.75 (2H, s, H3, H5) ppm.

4.1.7.6. 3-(3-Trifluoromethyl-4-(3-(3-methylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5- trifluoromethyl-1,2,4-oxadiazole 10m

White solid, yield 22 %, mp 102–104 °С (iPrOH). Mass (EI), m/z (I_relat.(%)): 464.3187 [M]⁺ (73). C₁₈H₁₄F₆N₄O₄. ¹H NMR (DMSO-d₆): δ 2.17 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.77 (3H, d, J = 2.4, NCH₃), 2.98 (2H, t, J = 7.6, CH₂CH₂CH₂O), 4.30 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.60 (1H, s, isoxazole), 7.51 (1H, d, J = 8.0, H6), 8.24 (1H, s, H3), 8.32 (d, 1H, J = 8.0, H5), 8.61 (1H, brs, NH) ppm.

4.1.8. General procedure for the synthesis of compounds 10f–l, 13, 20

A mixture of 6c–i or 11 or 19 (1 mmol) and dimethylamine solution 33 wt. % in H₂O or 17 wt. % in dioxane (5 mmol) was heated at 50–60 °C for 1–12 h. The cooled reaction mixture was concentrated in vacuo. The residue was treated by water and stored in the refrigerator for 12 h. Crystals were collected and recrystallized from the corresponding solvent.

4.1.8.1. 3-(4-(3-(3-N,N-dimethylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4- oxadiazole 10f

White solid, yield 60 %, mp 95–98 °С (EtOH). Mass (EI), m/z (I_relat.(%)): 410.3473 [M]⁺ (39). C₁₈H₁₇F₃N₄O₄. ¹H NMR (DMSO-d₆): δ 2.15 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.95 (3H, s, NCH₃), 3.00 (2H, t, J = 7.6, CH₂CH₂CH₂O), 3.05 (3H, s, NCH₃), 4.20 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.50 (1H, s, isoxazole), 7.20 (2H, d, J = 7.50, H2, H6), 8.00 (2H, d, J = 7.5, H3, H5) ppm.

4.1.8.2. 3-(3-Methyl-4-(3-(3-N,N-dimethylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5- trifluoromethyl-1,2,4-oxadiazole 10g

White solid, yield 70 %, mp 88–89 °С (EtOH/H₂O). Mass (EI), m/z (I_relat.(%)): 424.3739 [M]⁺ (37). C₁₉H₁₉F₃N₄O₄. ¹H NMR (DMSO-d₆): δ 2.22 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.24 (3H, s, CH₃Ph), 3.00 (3H, s, NCH₃), 3.05 (2H, t, J = 7.6, CH₂CH₂CH₂O), 3.10 (3H, s, NCH₃), 4.20 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.50 (1H, s, isoxazole), 7.20 (1H, d, J = 8.8, H6), 7.80 (1H, d, J = 1.5, H5), 7.81 (1H, dd, J = 1.5, 8.8, H3) ppm.

4.1.8.3. 3-(3-Methoxy-4-(3-(3-N,N-dimethylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5- trifluoromethyl-1,2,4-oxadiazole 10h

White solid, yield 53 %, mp 92–94 °С (EtOH/H₂O). Mass (EI), m/z (I_relat.(%)): 440.3733 [M]⁺ (94). C₁₉H₁₉F₃N₄O₅. ¹H NMR (DMSO-d₆): δ 2.18 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.98 (3H, s, NCH₃), 3.00 (2H, t, J = 7.6, CH₂CH₂CH₂O), 3.11 (3H, s, NCH₃), 3.85 (3H, s, OMe), 4.20 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.50 (1H, s, isoxazole), 7.23 (1H, d, J = 8.8, H6), 7.51 (1H, d, J = 1.5, H3), 7.65 (1H, dd, J = 1.5, 8.8, H5) ppm.

4.1.8.4. 3-(3-Nitro-4-(3-(3-N,N-dimethylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5- trifluoromethyl-1,2,4-oxadiazole 10i

White solid, yield 37%, mp 62–64 °С (EtOH). Mass (EI), m/z (I_relat.(%)): 455.3449 [M]⁺ (52). C₁₈H₁₆F₃N₅O₆. ¹H NMR (DMSO-d₆): δ 2.20 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.98 (3H, s, NCH₃), 3.00 (2H, t, J = 7.6, CH₂CH₂CH₂O), 3.05 (3H, s, NCH₃), 4.40 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.51 (1H, s, isoxazole), 7.55 (1H, d, J = 8.8, H6), 8.32 (1H, dd, J = 1.5, 8.8, H5), 8.50 (1H, d, J = 1.5, H3) ppm.

4.1.8.5. 3-(3-Fluoro-4-(3-(3-N,N-dimethylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5- trifluoromethyl-1,2,4-oxadiazole 10j

White solid, yield 42 %, mp 83–85 °С (hexane). Mass (EI), m/z (I_relat.(%)): 428.3378 [M]⁺ (29). C₁₈H₁₆F₄N₄O₄. ¹H NMR (DMSO-d₆): δ 2.21 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.99 (3H, s, NCH₃), 3.10 (3H, s, NCH₃), 3.05 (2H, t, J = 7.6, CH₂CH₂CH₂O), 4.25 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.52 (1H, s, isoxazole), 7.40 (1H, t, J = 8.3, H3), 7.80–7.90 (2H, m, H5, H6) ppm.

4.1.8.6. 3-(3-Trifluoromethyl-4-(3-(3-N,N-dimethylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5- trifluoromethyl-1,2,4-oxadiazole 10k

White solid, yield 71 %, mp 160–162 °С (iPrOH). Mass (EI), m/z (I_relat.(%)): 478.3453 [M]⁺ (68). C₁₉H₁₆F₆N₄O₄. ¹H NMR (DMSO-d₆): δ 2.17 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.93 (3H, s, NCH₃), 2.98 (2H, t, J = 7.6, CH₂CH₂CH₂O), 3.05 (3H, s, NCH₃), 4.29 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.52 (1H, s, isoxazole), 7.54 (1H, d, J = 8.0, H6), 8.20 (1H, s, H3), 8.34 (1H, d, J = 8.0, H5) ppm.

4.1.8.7. 3-(3-Methoxy-5-nitro-4-(3-(3-N,N-dimethylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5- trifluoromethyl-1,2,4-oxadiazole 10l

White solid, yield 92 %, mp 76–78 °С (EtOH/H₂O). Mass (EI), m/z (I_relat.(%)): 485.3709 [M]⁺ (49). C₁₉H₁₈F₃N₅O₇. ¹H NMR (DMSO-d₆): δ 2.13 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.97 (3H, s, NCH₃), 3.00 (2H, t, J = 7.6, CH₂CH₂CH₂O), 3.03 (3H, s, NCH₃), 4.00 (3H, s, OMe), 4.25 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.50 (1H, s, isoxazole), 7.80 (1H, d, J = 2.0, H6), 8.07 (1H, d, J = 2.0, H4) ppm.

4.1.8.8. 3-(3-Amino-4-(3-(3-N,N-dimethylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5- trifluoromethyl-1,2,4-oxadiazole 13

Light-brown solid, yield 54 %, mp 84–85 °С (EtOH). Mass (EI), m/z (I_relat.(%)): 425.3618 [M]⁺ (59). C₁₈H₁₈F₃N₅O₄. ¹H NMR (DMSO-d₆): δ 2.18 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.99 (3H, s, NCH₃), 3.00 (2H, t, J = 7.6, CH₂CH₂CH₂O), 3.05 (3H, s, NCH₃), 4.15 (2H, t, J = 6.5, CH₂CH₂CH₂O), 5.15 (2H, brs, NH₂), 6.50 (1H, s, isoxazole), 6.95 (1H, d, J = 8.0, H6), 7.25 (1H, d, J = 8.0, H5), 7.40 (1H, s, H3) ppm.

4.1.8.9. 3-(4-(3-(3-N,N-dimethylcarbamoyl-isoxazol-5-yl)propoxy)pyridin-3-yl)-5-trifluoromethyl- 1,2,4-oxadiazole 20

White solid, yield 25 %, mp 74–75 °С (hexane). Mass (EI), m/z (I_relat.(%)): 411.3352 [M]⁺ (49). C₁₇H₁₆F₃N₅O₄. ¹H NMR (DMSO-d₆): δ 2.18 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.95 (2H, t, J = 7.6, CH₂CH₂CH₂O), 3.00 (3H, s, NCH₃), 3.05 (3H, s, NCH₃), 4.45 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.51 (1H, s, isoxazole), 7.02 (1H, d, J = 7.8, H3), 8.27 (1H, dd, J = 1.5, 7.8, H4), 8.79 (1H, d, J = 1.5, H6) ppm.

4.1.9. Synthesis of 3-(3-amino-4-(3-(3-carbetoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl- 1,2,4-oxadiazole 11

A mixture compound 6f (1 mmol) and SnCl₂·2H₂O (5 mmol) in ethanol was stirred for 12 h at room temperature. The reaction mixture was diluted with ammonia water solution and extracted with ethyl acetate (3-times). The combined organic phases were washed with water, dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was treated by water and stored in the refrigerator for 2 h. Crystals were collected and recrystallized from ethanol. White solid, yield 93%, mp 56–58 °С (EtOH). Mass (EI), m/z (I_relat.(%)): 426.3466 [M]⁺ (61). C₁₈H₁₇F₃N₄O₅. ¹H NMR (DMSO-d₆): δ 1.31 (3H, t, J = 7.1, CH₃CH₂O), 2.17 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 3.05 (2H, t, J = 7.6, CH₂CH₂CH₂O), 4.10 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.35 (2H, q, J = 7.1, CH₃CH₂O), 5.10 (2H, brs, NH₂), 6.74 (1H, s, isoxazole), 6.98 (1H, d, J = 8.0, H6), 7.25 (1H, d, J = 8.0, H5), 7.35 (1H, s, H3) ppm.

4.1.10. General procedure for the synthesis of compounds 12a and 14a

A solution of 11 or 13 (1 mmol) and acetic anhydride (0.04 mol) was heated at 80 °C for 2–2.5 h. The cooled reaction mixture was treated by cold water and stirred for 1–2 h. The precipitate was collected by filtration and recrystallized from the corresponding solvent.

4.1.10.1. 3-(3-Acetylamino-4-(3-(3-carbethoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl- 1,2,4-oxadiazole 12a

White solid, yield 90 %, mp 132–134 °С (EtOH). Mass (EI), m/z (I_relat.(%)): 468.3833 [M]⁺ (61). C₂₀H₁₉F₃N₄O₆. ¹H NMR (DMSO-d₆): δ 1.32 (3H, t, J = 7.1, CH₃CH₂O), 2.10 (3H, s, CH₃CO), 2.25 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 3.04 (2H, t, J = 7.6, CH₂CH₂CH₂O), 4.25 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.35 (2H, q, J = 7.1, CH₃CH₂O), 6.60 (1H, s, isoxazole), 7.25 (1H, d, J = 8.0, H6), 7.85 (1H, d, J = 8.0, H5), 8.80 (1H, s, H3), 9.20 (1H, s, NH) ppm.

4.1.10.2. 3-(3-Acetylamino-4-(3-(3-N,N-dimethylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5- trifluoromethyl-1,2,4-oxadiazole 14a

White solid, yield 34 %, mp 135–137 °С (hexane/EtOAc). Mass (EI), m/z (I_relat.(%)): 467.3985 [M]⁺ (60). C₂₀H₂₀F₃N₅O₅. ¹H NMR (DMSO-d₆): δ 2.20 (3H, s, CH₃CO), 2.26 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.98 (3H, s, NCH₃), 3.00 (2H, t, J = 7.6, CH₂CH₂CH₂O), 3.10 (3H, s, NCH₃), 4.23 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.52 (1H, s, isoxazole), 7.26 (1H, d, J = 8.0, H6), 7.75 (1H, d, J = 8.0, H5), 8.75 (1H, s, H3), 9.20 (1H, brs, NH) ppm.

4.1.11. General procedure for the synthesis of compounds 12b and 14b

To a solution of 11 or 13 (1 mmol) in acetic acid was added a solution of 2,5-dimethoxyfuran (1.1 mmol) in acetic acid drop-by-drop at room temperature and then the mixture was stirred at 50 °C for 12–16 h. The cooled reaction mixture was treated by cold water, stirred for 1 h and stored in the refrigerator for 12 h. The precipitate was collected by filtration and recrystallized from the corresponding solvent.

For compound 14b, the cooled reaction mixture was treated by cold water and extracted with ethyl acetate (3-times). The combined organic phases were washed with water, dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by column chromatography with chloroform as eluent. The product fractions were concentrated in vacuo. Compound 14b was obtained as oil.

4.1.11.1. 3-(3-(1H-Pyrrol-1-yl)-4-(3-(3-carbethoxy-isoxazol-5-yl)propoxy)phenyl)-5- trifluoromethyl-1,2,4-oxadiazole 12b

Light-brown solid, yield 17 %, mp 95–97 °С (EtOH/H₂O). Mass (EI), m/z (I_relat.(%)): 476.4053 [M]⁺ (47). C₂₂H₁₉F₃N₄O₅. ¹H NMR (DMSO-d₆): δ 1.27 (3H, t, J = 7.1, CH₃CH₂O), 2.17 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.95 (2H, t, J = 7.6, CH₂CH₂CH₂O), 4.20 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.35 (2H, q, J = 7.1, CH₃CH₂O), 6.25 (2H, m, H3’,H4’), 6.55 (1H, s, isoxazole), 7.20 (2H, m, H2’, H5’), 7.50 (1H, d, J = 8.0, H6), 8.00 (1H, d, J = 8.0, H5), 7.90 (1H, s, H3) ppm.

4.1.11.2. 3-(3-(1H-Pyrrol-1-yl)-4-(3-(3- N,N-dimethylcarbamoyl-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 14b

Colorless oil, yield 40 %. Mass (EI), m/z (I_relat.(%)): 475.4205 [M]⁺ (52). C₂₂H₂₀F₃N₅O₄. ¹H NMR (DMSO-d₆): δ 2.17 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.93 (2H, t, J = 7.6, CH₂CH₂CH₂O), 3.00 (3H, s, NCH₃), 3.11 (3H, s, NCH₃), 4.24 (2H, t, J = 6.5, CH₂CH₂CH₂O), 6.25 (2H, m, H3’, H4’), 6.50 (1H, s, isoxazole), 7.20 (2H, m, H2’, H5’), 7.49 (1H, d, J = 8.0, H6), 7.94 (1H, s, H3), 8.01 (1H, d, J = 8.0, H5) ppm.

4.1.12. Synthesis of 3-(3-(((dimethylamino)methylene)amino)-4-(3-(3-carbethoxy-isoxazol-5-yl)propoxy)phenyl)-5-trifluoromethyl-1,2,4-oxadiazole 12c

A solution of 11 (1 mmol) and DMF-DMA (1.4 mmol) in ethanol was refluxed for 3.5–4 h. The cooled reaction mixture was treated by cold water and stirred for 12 h. The solid was collected and recrystallized from hexane. White solid, yield 13 %, mp 79–82 °С. Mass (EI), m/z (I_relat.(%)): 481.4251 [M]⁺ (45). C₂₁H₂₂F₃N₅O₅. ¹H NMR (DMSO-d₆): δ 1.32 (3H, t, J = 7.1, CH₃CH₂O), 2.12 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.98 (3H, s, NCH₃), 3.00 (3H, s, NCH₃), 3.05 (2H, t, J = 7.6, CH₂CH₂CH₂O), 4.10 (2H, t, J = 6.5, CH₂CH₂CH₂O), 4.35 (2H, q, J = 7.1, CH₃CH₂O), 6.74 (1H, s, isoxazole), 7.18 (1H, d, J = 8.0, H6), 7.56 (1H, d, J = 8.0, H5), 7.45 (1H, s, H3), 7.75 (1H, s, NCH) ppm.

4.1.13. Synthesis of 6-(pent-4-yn-1-yloxy)nicotinonitrile 16

A mixture 6-chloronicotinonitrile 15 (1 mol), 4-pentyn-1-ol (1.2 mol), finely divided K₂CO₃ (2 mol) in DMF was heated at 100 °C for 8–9 h. The cooled mixture was diluted with water and extracted with ethyl acetate (3-times). The combined organic phases were washed with water, dried over anhydrous Na₂SO₄ and concentrated in vacuo to an oil, which was dissolved in CHCl₃, filtered though a short column of Silicagel 60, and concentrated in vacuo. The residue was treated by water and stored in the refrigerator for 2 h. Crystals were filtered off and recrystallized from ethanol. Yellow solid, yield 47 %, mp 55–57 °C. Mass (EI), m/z (I_relat.(%)): 186.2099 [M]⁺ (45). C₁₁H₁₀N₂O. ¹H NMR (DMSO-d₆): 1.99 (2H, dt, J = 6.5, 7.6, CH₂CH₂CH₂O), 2.26 (2H, t, J = 7.6, CH₂CH₂CH₂O), 2.75 (1H, s, CHCCH₂), 4.26 (2H, t, J = 6.5, CH₂CH₂CH₂O), 7.00 (1H, d, J = 7.8, H3), 8.01 (1H, dd, J = 1.5, 7.8, H4), 8.52 (1H, d, J = 1.5, H6) ppm.

4.2. In vitro antiviral testing

4.2.1. Compounds

Stock solutions (10,000 μM) of test and reference compounds (pleconaril and guanidine hydrochloride) were prepared in DMSO and stored at 4 °C until use in cytotoxicity and antiviral studies.

4.2.2. Cells and viruses

HeLa Ohio (human cervix carcinoma; FlowLabs) were used in cytotoxic and anti-viral studies as well. HeLa cells were grown in Eagle’s minimal essential medium (MEM/E; Cambrex, Verviers, Belgium) supplemented with 10% fetal calf serum (FCS: HeLa Ohio, PAA, Pasching, Austria). The medium in cytotoxicity and antiviral studies (test medium) contained only 2% of FCS.

Virus stocks of the coxsackievirus B3 prototype strain Nancy (CVB3 Nancy; Chumakov Institute of Poliomyelitis and Viral Encephalitides, Moscow, Russia), the clinical coxsackievirus B3 isolate 97927 (Robert Koch Institute, Berlin, Germany), the three pleconaril-resistant mutants of coxsackievirus B3 97927 [17], rhinoviruses A2, B5 (kindly provided by Dr. J. Seipelt, Greenhills Biotechnology, Ltd., Vienna, Austria), rhinovirus B14 (kindly provided by Dr. H.-P. Grunert, Universitatsklinikum Benjamin Franklin, Berlin, Germany) were prepared by incubating confluent HeLa cell monolayers (FlowLabs) grown in cell culture flasks with a certain amount of virus at 37 °C (coxsackieviruses B3 and rhinovirus B14) or 33°C (rhinoviruses A2 and B5) and 5% CO₂. When a nearly complete cytopathic effect became microscopically visible, infected cells were frozen at − 80 °C and thawed three times, the cell debris was removed by centrifugation, and aliquots of the virus-containing supernatant were stored as virus stock at −80 °C until use. Titers of virus stocks were determined by endpoint dilution in HeLa cells according to Reed and Muench [36].

4.2.3. Determination of cytotoxicity

To determine the 50% cytotoxic concentration (CC₅₀), 2-day-old confluent HeLa cell monolayer grown in the internal 60 wells of 96-well plates were incubated with serial half-log dilutions of compounds (100 μM, 31.6 μM, 10 μM, 3.16 μM etc.) in test medium for 72 h (37 °C, 5% CO₂) in test medium or test medium without compound (six mock-treated cell controls). After an incubation time of 72 h, the cells were fixed and stained with a crystal violet formalin solution as described previously [21]. The crystal violet-stained monolayers were treated for 20 min with 100 μL of lysis buffer (0.8979 g of sodium citrate and 1.25 mL of 1N HCl in 98.05 mL 47.5 % ethanol) to elute the crystal violet. After dye elution, the optical density of individual wells was quantified spectrophotometrically at 550/630 nm with a microplate reader (Dynex, Guernsey, GB). Cell viability of individual compound-treated wells was evaluated as the percentage of the mean value of optical density resulting from the six mock-treated cell controls which was set 100% cell viability. The 50% cytotoxic concentration (CC₅₀) was defined as the compound concentration reducing the mean viability of cell controls by 50%. Mean CC₅₀ values were calculated from at least 3 tests.

4.2.4. Cytopathic effect (CPE)-inhibition assay

The CPE-inhibition assays have been performed as described previously with some modifications [21]. Briefly, the tests were carried out in 1-day-old (all rhinoviruses) or 2-day-old (all coxsackieviruses B3) HeLa cell monolayers growing in the inner 60 wells of 96-well flat-bottomed microtiter plates. After removal of culture medium, 50 μL of compound solution (serial half-log dilutions of compounds: 100 μM, 31.6 μM, 10 μM, 3.16 μM etc. in test medium) and 50 μL of a virus suspension with a certain multiplicity of infection were added to the cells. The multiplicity of infection was 0.015–0.028 for coxsackievirus B3 97927, 0.015–0.02 for coxsackievirus B3 97927 I1207K/I1092, 0.002–0.0025 for coxsackievirus B3 97927 I1207M/I1092, 0.002–0.0025 for coxsackievirus B3 97927 I1207M/I1092, 0.01–0.015 for coxsackievirus B3 97927 I1207T/I1092, 0.002 for coxsackievirus B3 Nancy, 0.03 for rhinovirus A2, 0.01–0.04 for rhinovirus B5, and 0.005 for rhinovirus B14. Six wells of non-infected and six wells of infected cells without the test compound served as cell and virus control, respectively, on each plate. Guanidine hydrochloride was included as positive control compounds for all CVB3. With the exception of rhinovirus B5 (Pleconaril resistant), pleconaril was used as reference compound for rhinoviruses. Serial dilutions of test compounds were included in each assay run for assay validation. Using the crystal violet uptake assay described for cytotoxic investigations, the inhibition of the virus-induced CPE was scored 48 h (all coxsackieviruses B3) or 72 h (all rhinoviruses) post infection when untreated infected control cells showed maximum cytopathic effect and the positive control compound-treated wells a 50% or 100% protection. The percentage of antiviral activity of the tests compounds were calculated according to Pauwels et al. [26]. The mean IC₅₀ values of the antiviral active compounds were calculated from at least 3 separate experiments.

4.3. Pharmacokinetic study in mice

All animal procedures were approved by the Institutional Animal Care and Use Committee of Central Research Institute of Tuberculosis (Moscow, Russia) and conducted in accordance with the state industry standards GOST 33215–2014 and GOST 33216–2014 (in harmonization to the European Directive 2010/63/ЕС).

4.3.1. Experimental animals

Twenty two white Balb/C male mice with average body weight of 20 g were selected for the study. All the test animals had free access to water but were deprived of food for 1 hour before administration of the compound. This regimen was continued for another hour after the administration.

4.3.2. Compound administration and sample collection and preparation

Compound in a dose of 100 mg/kg of body weight was administered as 0.5 ml of prepared suspension in 0.5% CMC with one drop Tween-80 by intragastric intubation. The animals were euthanized by decapitation for blood and brain sampling. Blood and brain samples (3 animals per time point) were taken at 0.5, 1, 2, 3, 5 and 7 h after administration for pharmacokinetic evaluation. Blood was collected in heparinized tubes and centrifuged at 3500 RPM for 15 minutes. Plasma was separated from formed elements and immediately frozen at −20 °C in freezer. This storage continued before transferring of plasma for analysis. Serum from 6 untreated animals was used as control and for equipment calibration with compound. Mice brain was immediately frozen at −120 °C. A mixture of 50 μL of plasma and 150 μL of MeCN by intensive shaking (Vortex) for 30 sec. Centrifugation 14000g for 4 min. After centrifugation the 150 μL of organic layer was injected into the HPLC column.

4.3.3. HPLC-MS Conditions

Chromatographic separation was achieved on an Agilent Eclipse XDB-C18 column (4.6 mm × 100 mm, 3.5 μm) using an Agilent 1290 Infinity HPLC system equipped with the binary pump, auto-sampler, column oven and degasser. The mobile phase containing 0.1% formic acid in acetonitrile (A) and 0.2% formic acid in water (B) was at a flow rate of 0.4 mL/min. The gradient program was as follows: 0.0–3.0 min, 60% B (separation); 3.0–4.0 min, 60 → 97% B (washing and regeneration); 4.0–6.0 min, 97% B; 6.0–6.1 min, 97 → 60% B; 6.1–9.0 min, 60% B. The separation temperature was set at 40 °C and the sample injection volume was 2 μL.

Mass detection was operated on an Agilent 6460 triple quadrupole mass spectrometer coupled with electrospray ionization (ESI) source. The mass spectrometer was set in positive ion mode. The optimum MS parameters were maintained as follows: nebulizer, 35 psi; drying gas (N₂) flow rate, 12 L/min; capillary temperature, 350 °C.

4.3.4. Calibration

The calibration curve was based on the model serum (45 μL of serum + 5 μL of standard solution). Five levels of concentrations were used: 50 ng/mL, 250 ng/mL, 500 ng/mL, 1000 ng/mL, 5000 ng/mL.

4.3.5. Calculation of pharmacokinetic parameters

Pharmacokinetic parameters were calculated with the ESTRIP computer program using the model-independent method. The area under the plasma concentration-time curve from time zero to infinity (AUC_0-∞) was calculated by the trapezium method with extrapolation to time infinity. The maximum concentration C_max and the time when it occurred (T_max) were obtained by visual inspection of the pharmacokinetic curve. The total clearance CL was calculated as dose/AUC_0-∞. The volume of distribution V_d was calculated as CL/k_el, where k_el is the elimination constant. The half-life T_1/2 was calculated using equation ln(2)/k_el = 0.693/k_el. The mean drug retention time in systemic bloodstream MRT was calculated by AUMC/AUC, where AUMC is the area under the first moment curve.

4.4. CYP3A4 Induction

Compound 10g and pleconaril were tested in cryopreserved hepatocytes (BioIVT, Westbury, NY) (at 1, 10, 100 μM) using 10 μM rifampicin as positive control and midazolam as the substrate for the enzyme (Eurofins Panlabs, Inc. St. Charles MO).

4.5. SOS chromotest

The SOS chromotest experiment was reported in [37]. In brief, Escherichia coli PQ37 strain was grown overnight at 37 °C, with shaking, in Luria Broth Base supplemented with 50 μg/mL Ampicillin. Bacteria were grown to mid-logarithmic phase and adjusted to an optical density of 0.4. The second day, Luria Broth Base supplemented with 1.5% Agar was autoclaved for 15 minutes at 121 °C and then put into a water bath at 55 °C for 1 hour. 80 mL of warm agar was transferred into a plastic bottle (Milian, PETG 2019–0125) supplemented with 50 mg/mL ampicillin and 0.005% X- Gal (dissolved into DMF). 4 mL of E. coli PQ37 was added to the mixture and 70 mL of it was transferred into a square petri dish (Grenier, 120 × 120 mm). The Petri dish containing the mixture was dried 15 minutes at RT and once the mixture became solid, 6 mm absorbent disks were stuck on the plate. 5 μL of each compound were added on the disk with a pipetman (Gilson P20) and the petri dish was incubated overnight at 37 °C. The third day, zones of inhibition were recorded with a ruler and a picture of the plate was taken with a scanner (Canon 8800F). The zone of inhibition was calculated by eye using a ruler and presence/absence of blue halo was reporter.

Fig 1.

Structures of well-known antipicornaviral drug candidates, pleconaril and vapendavir (A) and our structure-activity relationship (SAR) study of pleconaril-like compounds described in this article (B)

Highlights.

• A series of novel pleconaril derivatives were synthesized and evaluated.

• Analogues with monosubstitution in the phenyl ring are more active than with disubstitution.

• Dimethylamido group in isoxazole ring is critical for inhibition of pleconaril-resistant viruses.

• Compound 10g is the most potent antienteroviral compound in this series.

• This derivative also show good bioavailability in mice.