key: cord-033493-kslzdy8q
authors: Hebishy, Ali M. S.; Salama, Hagar T.; Elgemeie, Galal H.
title: New Route to the Synthesis of Benzamide-Based 5-Aminopyrazoles and Their Fused Heterocycles Showing Remarkable Antiavian Influenza Virus Activity
date: 2020-09-21
journal: ACS Omega
DOI: 10.1021/acsomega.0c02675
sha: 
doc_id: 33493
cord_uid: kslzdy8q

[Image: see text] This study describes a new route to the synthesis of novel benzamide-based 5-aminopyrazoles and their corresponding pyrazolo[1,5-a]pyrimidine and pyrazolo[5,1-c][1,2,4]triazine derivatives. Benzamide-based 5-aminopyrazoles were prepared through a reaction of benzoyl isothiocyanate with malononitrile in KOH–EtOH followed by alkylation with alkyl halides and then a reaction with hydrazine. In an attempt to react benzoyl isothiocyanate with ethyl cyanoacetate in KOH–EtOH followed by alkylation with methyl iodide at room temperature and then a reaction with hydrazine has resulted in the formation of 3-ethoxy-5-phenyl-1H-1,2,4-triazole. The structures of the new compounds were characterized by mass spectroscopy, (1)H nuclear magnetic resonance ((1)H NMR) spectroscopy, infrared spectroscopy (IR), and X-ray analysis. The new compounds were tested in vitro for their anti-influenza A virus (subtype H5N1) activity. Among the synthesized compounds, eight compounds 3b, 4, 10b, 10c, 12a, 19, 21a, and 21b were found to possess significant antiviral activities against bird flu influenza (H5N1) with viral reduction in the range of 85–65%.

The emergence of bird flu virus (H5N1) 23 years ago has caused several diseases in mankind over the past few years, and now, it is spreading in the world a very deadly influenza pandemic, which is Covid-19 and has caused the death of many people. We had to put in place several measures to prevent the spread of these influenza viruses by developing pharmaceutical strategies to design drugs for combating influenza viruses of all kinds. 5-Aminopyrazoles and their fused triazine and pyrimidine ring systems are analogs of purines and thioguanines, which are the mostly used antimetabolic agents. Pyrazoles share their structural design with several drugs, and biologically dynamic compounds exhibit activities such as anti-inflammatory, anticancer, and antihypertensive. 1−3 Our group has actively embarked on a program for the development of new methods for the synthesis of 5-aminopyrazoles as potential bioactive agents. 4,5 5-Aminoyrazoles have been used to synthesize numerous other fused heterocycles. Pyrazolo [1,5-a] pyrimidines, in particular, have shown valuable pharmaceutical uses including various antiviral, antimicrobial, and antitumor activities. 6−11 Some compounds comprising this scaffold are official and commercialized drugs, for example, zaleplon (A), lorediplon (B), indiplon (C), anagliptin (D), and ocinaplon (E) (Figure 1 ). We have recently reported different successful synthetic methods to prepare pyrazolo [1,5-a] pyrimidines, which found application and appear to constitute new classes of antimetabolic agents. 12−15 Pyrazolo [5,1-c] triazines Synthesis of 3-ethoxy-5-phenyl include a large number of compounds with broad biological activities. The pyrazolo [5,1-c] triazines are well known as the most potent scaffold for synthesizing potential therapeutics. The applications of these ring systems as kinase inhibitors are growing and are very successful in the last few years. 16−19 We have recently reported different successful synthetic methods to prepare pyrazolotriazines, which found application and appear to constitute new classes of antimetabolic agents. 20, 21 One of our previously reported series of novel 5-aminopyrazoles F and G ( Figure 2 ) was used by others as a starting material for the synthesis of pyrazolopyrimidines. 22−27 Studies revealed that 5-aminopyrazoles act as a building block for various functionalized pyrazolopyrimidines as purine analogs. 28−37 These interesting results have promoted our research group to explore other synthetic methods for the preparation of 5-aminopyrazoles for synthesizing pyrazolotriazines and pyrazolopyrimidines and investigating their use as antiviral agents in chemotherapy. In light of these results and as part of our program directed toward the preparation of potential antiviral antibiotics, the present paper deals with a novel synthesis of novel benzamide-based 5-aminopyrazoles and their use in synthesizing pyrazolo- [5,1-c] triazine and pyrazolo[1,5-a]pyrimidine ring systems using innovative synthetic approaches.

Chemistry. It has been found that benzoyl isothiocyanate reacted with malononitrile in potassium hydroxide−ethanol with heating to give the corresponding stable potassium 2cyano-ethylene-1-thiolate salt 2. 5-Aminopyrazole 4 was prepared by alkylation of the potassium 2-cyano-ethylene-1thiolate salt 2 with an alkyl halide at room temperature to offer N-(2,2-dicyano-1-(alkylthio)vinyl)benzamide 3 followed by a reaction with hydrazine hydrate by refluxing ethanol containing a catalytic amount of piperidine (Scheme 1). The structures of 3 and 4 were established on the basis of their elemental analysis and spectral data (IR, 13 C NMR, 1 H NMR, and MS). Structure 4 was confirmed by its mass (m/z 227), which agrees with its molecular formula C 11 H 9 N 5 O. The 1 H NMR spectrum of compound 4 revealed a broad singlet at 5.37 ppm for the NH 2 group, a multiplet at a range of 7.50−8.09 ppm for the phenyl moiety, and two broad singlets at 11.73 and 12.33 ppm assigned to two NH groups.

The reactivity of 5-aminopyrazole derivative 4 to form the diazonium salt 5 was studied, and the latter was coupled with malononitrile or ethyl cyanoacetate in sodium acetate/EtOH to give the corresponding pyrazolotriazines 6a,b (Scheme 2).

The structures of the resultant N-(pyrazolo [5,1-c] [1, 2, 4 ]triazin-7-yl)benzamides 6a,b were confirmed according to their spectral data. Thus, the 1 H NMR spectrum for compound 6a revealed a multiplet at a range of 7.46−8.19 ppm for the presence of the phenyl group, a broad singlet at 3.60 ppm for the NH 2 group, and a broad singlet at 10.47 ppm for an NH group.

It was found that the 5-aminopyrazole 4 reacts with the sodium salts of (hydroxymethylene)cycloalkanones 7a, b in the presence of piperidine acetate−acetic acid to give an adduct, where the structure of 8a, b was established. The reaction begins with an initial nucleophilic attack from the external amino group to the formyl group followed by cyclization and then removal of one molecule of water to produce the angular tricyclic compounds 8a,b. In the presence of an acid medium, first, the protonation of the ring nitrogen occurs, which is the most nucleophilic center 38−43 in compound 4, which directs the exocyclic amino group to attack the unhindered formyl group of 7 to yield compounds 8a,b (Scheme 3). The 1 H NMR spectrum of 8a showed the existence of a signal at δ 8.80 ppm assigned to a pyrimidine-H proton.

The 5-aminopyrazole 4 reacted with arylmethylene malononitriles 9 with piperidine as a catalyst to give the pyrazolo[1,5-a]pyrimidines 10a−c. The reaction proceeded by Michael addition of the exocyclic amino group of 4 to the double bond of 9 followed by cyclization through the addition of the ring NH to the cyano group to give the pyrazolo[1,5a]pyrimidines 10 (Scheme 3). The structures of 10a−c were confirmed by 1 H NMR, which revealed for compound 10b a singlet at 3.88 ppm assigned to the OCH 3 group, a singlet at 9.18 ppm assigned to the NH 2 group, a multiplet at a range of 7.15−8.23 ppm for aromatic protons, and a broad singlet at 12.19 ppm to indicate the presence of the NH group.

The reactivity of diketones and keto esters such as acetyl acetone 11a and ethyl acetoacetate 11b with 5-aminopyrazole 4 was studied through a reaction with piperidine and boiling ethanol to afford the corresponding pyrazolo[1,5-a]pyrimidines 12a,b.

An attempt to react benzoyl isothiocyanate with ethyl cyanoacetate in KOH−EtOH with heating followed by alkylation with methyl iodides at room temperature has resulted in the formation of the corresponding (E)-ethyl 3benzamido-2-cyano-3-(methylthio)acrylate 14. The reaction of (E)-ethyl 3-benzamido-2-cyano-3-(methylthio)acrylate 14 with hydrazine was investigated. The reaction between 14 and hydrazine gave a product whose mass spectra were not consistent with the proposed pyrazole structure 16, and other spectroscopic measurements did not allow us to unambiguously identify the product, and thus, the X-ray crystal structure was determined as shown in Figure 3 , 44 confirming the exclusive presence of the triazole derivative 19 as the sole product in the solid state. The formation of 19 from the reaction of 14 and hydrazine is suggested to proceed via initial addition of basic nitrogen in hydrazine to the double bond of 14 followed by the formation of the adduct 15 and elimination of ethyl cyanoacetate. The adduct 15 leads to the formation of the favored, kinetically and thermodynamically controlled 3ethoxy-5-phenyl-1H-1,2,4-triazole product 19 (Scheme 4). The 1 H NMR spectra of the product 19 revealed the presence of an ethoxy group as a broad singlet at δ1.37 ppm assigned to the CH 3 group and a broad singlet at δ 4.34 ppm assigned to the CH 2 group, a multiplet at δ 7.47−7.93 ppm assigned to the phenyl group, and a triazole ring NH at δ 13.72 ppm.

The potassium ethylenethiolate salts 2 and 13 reacted with tetra-O-acetyl-glucopyranosyl bromide 20 at room temperature and in ethanol to give with a high yield the corresponding Sglucosides 21a,b, respectively. The chemical structures of the prepared compounds 21a,b were confirmed by elemental analyses and spectroscopy ( 1 H NMR and IR) studies. For example, the 1 H NMR spectrum of compound 21b showed an anomeric proton at δ 6.10−6.12 ppm as a doublet, and the coupling constant (J 1′,2′ = 9.2 Hz) proved H-1′to be transdiaxial to H-2′. 45−50 The signals resonating at 3.92, 3.99, 4.04, 4.89, 5.03, and 5.61 ppm are assigned to six glucose protons, and the four singlets appearing from δ 1.59 ppm to 2.00 ppm are assigned to four acetyl groups. The signal for the C-1′ atom in the 13 C NMR spectrum of 21b appeared at δ 80.31, and the signals for C-6′, C-4′, C-2′, C-3′, and C-5′ appeared at δ 61.50, 68.76, 69.46, 73.47, and 79.82 ppm, respectively. An attempt to remove the protection groups at 21 by methanol−ammonia did not result in the formation of the corresponding free glycosides. The structure 21 was suggested to be present in the E form and not in the Z form, which was demonstrated by the reaction of compounds 21 with hydrazine at room temperature in piperidine−ethanol to give the corresponding 5-aminoprazole 4. The structure of 4 was confirmed on the basis of elemental analysis and spectral data. Antiviral Activity. The antiviral activity was measured for the synthesized compounds with respect to the H5N1 influenza virus strain A/Egypt/M7217B/2013 using MTT 50 ) and plaque reduction assays 52 exploring the cytotoxicity and inhibition percentage values, respectively. The anti-influenza drug zanamivir was used as a positive control. 53 Antiviral bioassay data (see Table 1 and Figure 4 ) indicated that most of the compounds demonstrated a dosedependent inhibition behavior. Based on the U.S. National Cancer Institute (NCI) and GERAN protocol, 54,55 the LD 50 values indicate that most tested compounds, especially compound 3b, are safe for healthy cells, while the plaque reduction assay showed that compounds 3b, 4, and 10b have higher therapeutic indices compared to the other synthesized compounds. In particular, compound 3b exhibited the highest antiviral activity at the given concentrations with percentage of virus reduction reaching 85% at a concentration of 0.25 μmol/ ml. On the other hand, compounds 3c, 10c, 12a, 19, 21a, and 21b demonstrated a moderate anti-H5N1 activity with viral inhibition over 65 percent. In general, it has been observed that compound 3b with an ethylthio group at a concentration of 0.25 μmol/ml was more active compared to 3a and 3c with methylthio and benzylthio groups, respectively. Additionally, conversion of N-(2,2-dicyano-1-(alkylthio)vinyl)benzamides 3a and 3c to the 5-aminopyrazole 4 relatively enhanced the activity at the same concentration. The presence of pyrazolotriazine moieties in compounds 6a, b decreases the activity compared to the 5-aminopyrazole 4. Compound 10b with a pyrazolo[1,5-a]pyrimidine scaffold and amino-, cyano-, and methoxy phenyl groups was found to have the highest potency in the series at a concentration of 0.125 μmol/ml (83%). Unexpectedly, the synthesis of derivatives (21a and 21b) with a sugar moiety had no significant effect on the antiviral activity (Scheme 5), where the study showed that N-(2,2-dicyano-1-(alkylthio)vinyl)benzamide 3b with S-ethyl seemed to be more active than the corresponding derivatives of acyclic thioglucosides 21a,b. Synthesis of N-(5-amino-3,6-dicyano-7-arylpyrazolo[1,5a]pyrimidin-2-yl)benzamides 10a−c. Method: a mixture of compound 4 (2.27 g, 0.01 mol) and benzylidene propane-dinitrile 9a (1.54 g, 0.01 mol), (4-methoxy benzylidene) propanedinitrile 9b (1.84 g, 0.01 mol), or (4-chlorobenzylidene) propanedinitrile 9c (1.88 g, 0.01 mol) was refluxed for 5 h in ethanol (20 mL) containing drops of piperidine. The reaction mixture was allowed to cool to room temperature. The precipitate was collected by filtration and crystallized from DMF.

N-(5-Amino-3,6-dicyano-7-phenylpyrazolo[1,5-a]pyrimidin-2-yl)benzamide 10a. Yellow powder; (DMF); yield 55%; mp>300°C; 1 Synthesis of (E)-Ethyl 3-benzamido-2-cyano-3-(methylthio)acrylate 14. Method: a mixture of ethyl cyanoacetate (1.13 g, 0.01 mol) and benzoyl isothiocyanate (1.63 g, 0.01 mol) was heated for 30 min in ethanol (25 mL) in the presence of potassium hydroxide (0.56 g, 0.01 mol). After cooling, methyl iodide (1 mmol) was added, and the mixture was stirred overnight. The resulting solid product was collected by filtration and recrystallized from ethanol.

MTT Cytotoxicity Assay (LD 50 ). The samples were diluted to 10-fold serially using the Dulbecco's modified Eagle's medium (DMEM). 51−56 Then, a preparation of test compound stock solutions was carried out in diluted DMSO with distilled water (10%). Cytotoxicity testing of the extracts was performed with Madin−Darby canine kidney (MDCK) cells using the method of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) 51 with some minor modifications. Cells were placed in 96-well plates and incubated for 1 day at room temperature in 5% CO 2 . After 1 day, the cells were treated with different concentrations of the tested compounds. The supernatant was discarded after further 24 h, and the cell monolayers were washed with a solution of phosphate saline (PBS). An MTT solution was added to each well and incubated at room temperature for 4 h, and then, a medium suction was applied. The formazine crystals formed were then dissolved in 200 μL of acidified isopropanol. The absorbance measurement of the formazine solutions at λ max of 540 nm with 620 nm as the reference wavelength was performed using a multiplate reader. Then, the determination of the cytotoxicity compared to the untreated cells was performed using the following equation. The plot of cytotoxicity percentage versus concentration of sample was used to calculate the concentration, which showed 50% cytotoxicity (LD 50 ). Plaque Reduction Assay. The plaque reduction assay was performed according to the method described by Hayden et al. 52 in a plate of six wells in which the MDCK cells (105 cells/ ml) were cultivated for 1 day at room temperature. 51−56 The virus A/CHICKEN/7217B/1/2013 (H5N1) was diluted to give 105 PFU/well and mixed with the tested samples and incubated for half hour at room temperature before being added to the cells. The growth medium was removed from the cell culture plates, and mixtures of virus−Cpd or virus−extract and virus−oseltamivir (100 μL/well) were inoculated (100 μL/well). After 1 hour of contact time for viral uptake, 3 mL of DMEM was added with 2% agarose on the monolayer cell, and platelets were left to harden and were incubated at room temperature until viral plaques form (3−4 days). Formalin (10%) was added for 2 h, and then, the plates were stained with 0.1% crystal violet in H 2 O. Control wells were included where untreated virus was incubated with MDCK cells, and at the end, plaque was counted and the percentage decrease in plaque formation compared to control wells was calculated as follows: The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c02675.

All spectral analysis data such as IR, 1 H NMR, and 13 C NMR spectra for the newly synthesized compounds (PDF)

Yellow powder; (ethanol); yield 53%; mp 64°C; 1 H NMR (400 MHz, DMSO-d 6 ): δ 1.36−1.41 (t, 3H, CH 3 ), 2.39 (s, 3H

Method: hydrazine hydrate (0,50 g, 0.01 mol) was added to a solution of (E)-ethyl 3-benzamido-2-cyano-3-(methylthio) acrylate (14) (2.90 g, 0.01 mol) in ethanol (20 mL) in the presence of drops of piperidine. The mixture was heated under reflux for 2 h and then poured onto ice. The solid product was filtered off, dried

Synthesis of acyclic thioglucosides 21a,b. Method: a mixture of malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) and benzoyl isothiocyanate (1.63 g, 0.01 mol) was heated for 10−20 min in ethanol

11 g, 0.01 mol) was added. The reaction mixture was stirred overnight at room temperature. The resulting solid was collected by filtration and was separated from traces of the reactant by preparative thinlayer chromatography using DCM as an eluent where compounds 21a

Yellow powder; yield 84%; mp>300°C; 1 H NMR (400 MHz, DMSO-d 6 ): δ 1.18−1.27 (t, 3H, CH2-CH 3 ), 1.59−2.00 (4 s, 12H

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