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. 2020 Sep 21;5(39):25104–25112. doi: 10.1021/acsomega.0c02675

New Route to the Synthesis of Benzamide-Based 5-Aminopyrazoles and Their Fused Heterocycles Showing Remarkable Antiavian Influenza Virus Activity

Ali M S Hebishy 1,*, Hagar T Salama 1, Galal H Elgemeie 1,*
PMCID: PMC7542596  PMID: 33043189

Abstract

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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, 1H nuclear magnetic resonance (1H 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%.

Introduction

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.

Figure 1.

Figure 1

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Pyrazolo[1,5-a]pyrimidine-based drugs A–D.

Figure 2.

Figure 2

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Structure of our previously reported 5-aminopyrazoles Fa,b and Ga,b.22−27

Results and Discussion

Chemistry

It has been found that benzoyl isothiocyanate reacted with malononitrile in potassium hydroxide–ethanol with heating to give the corresponding stable potassium 2-cyano-ethylene-1-thiolate salt 2. 5-Aminopyrazole 4 was prepared by alkylation of the potassium 2-cyano-ethylene-1-thiolate 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, 13C NMR, 1H NMR, and MS). Structure 4 was confirmed by its mass (m/z 227), which agrees with its molecular formula C11H9N5O. The 1H NMR spectrum of compound 4 revealed a broad singlet at 5.37 ppm for the NH2 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.

Scheme 1. Synthesis of N-(5-amino-4-cyano-1H-pyrazol-3-yl)benzamide 4.

Scheme 1

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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 1H 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 NH2 group, and a broad singlet at 10.47 ppm for an NH group.

Scheme 2. Synthesis of N-(pyrazolo[5,1-c][1,2,4]triazin-7-yl)benzamides 6a,b.

Scheme 2

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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 center38−43 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 1H NMR spectrum of 8a showed the existence of a signal at δ 8.80 ppm assigned to a pyrimidine-H proton.

Scheme 3. Synthesis of N-(pyrazolo[1,5-a]pyrimidin-2-yl)benzamides 8a,b, 10a–c, and 12a,b.

Scheme 3

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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[1,5-a]pyrimidines 10 (Scheme 3). The structures of 10a–c were confirmed by 1H NMR, which revealed for compound 10b a singlet at 3.88 ppm assigned to the OCH3 group, a singlet at 9.18 ppm assigned to the NH2 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 3-benzamido-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 3-ethoxy-5-phenyl-1H-1,2,4-triazole product 19 (Scheme 4). The 1H NMR spectra of the product 19 revealed the presence of an ethoxy group as a broad singlet at δ1.37 ppm assigned to the CH3 group and a broad singlet at δ 4.34 ppm assigned to the CH2 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.

Figure 3.

Figure 3

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X-ray single-crystal structure of the title compound 19 “Reproduced with permission of the International Union of Crystallography under the open-access license”.44

Scheme 4. Synthesis of 3-ethoxy-5-phenyl-1H-1,2,4-triazoles 19.

Scheme 4

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Reproduced with permission of the International Union of Crystallography under the open-access license.44

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 S-glucosides 21a,b, respectively. The chemical structures of the prepared compounds 21a,b were confirmed by elemental analyses and spectroscopy (1H NMR and IR) studies. For example, the 1H NMR spectrum of compound 21b showed an anomeric proton at δ 6.10–6.12 ppm as a doublet, and the coupling constant (J1′,2′ = 9.2 Hz) proved H-1′to be trans-diaxial 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 13C 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 cytotoxicity51 (LD50) and plaque reduction assays52 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 dose-dependent inhibition behavior. Based on the U.S. National Cancer Institute (NCI) and GERAN protocol,54,55 the LD50 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.

Table 1. Antiviral Activity of Tested Compounds Measured Using the Plaque Reduction Assay and MTT Cytotoxicity.

plaque reduction assays
  
comp. no. concentration μmol/ml initial viral counta viral counta inhibition % MTT assay LD50 (μmol/ml)a
PFU/mL
3a 0.125 0.250 6 × 106 6 × 106 5.0 × 106 4.0 × 106 16.67 33.33 200
3b 0.125 0.250 6 × 106 6 × 106 2.2 × 106 9.0 × 105 63.33 85 480
3c 0.125 0.250 6 × 106 6 × 106 2.8 × 106 2.1 × 106 53.33 65 200
4 0.125 0.250 6 × 106 6 × 106 2.0 × 106 1.4 × 106 66.67 76.67 30
6a 0.125 0.250 6 × 106 6 × 106 4.0 × 106 3.0 × 106 33.33 50 200
6b 0.125 0.250 6 × 106 6 × 106 3.0 × 106 3.2 × 106 50 46.67 100
8a 0.125 0.250 6 × 106 6 × 106 4.0 × 106 2.5 × 106 33.33 58.33 170
8b 0.125 0.250 6 × 106 6 × 106 b b b b 210
10b 0.125 0.250 6 × 106 6 × 106 1.0 × 106 b 83.33 b 140
10c 0.125 0.250 6 × 106 6 × 106 4.0 × 106 2.0 × 106 33.33 66.67 120
12a 0.125 0.250 6 × 106 6 × 106 4.6 × 106 2.0 × 106 23.33 66.67 210
12b 0.125 0.250 6 × 106 6 × 106 5.0 × 106 4.0 × 106 16.67 33.33 190
14 0.125 0.250 6 × 106 6 × 106 5.0 × 106 5.0 × 106 16.67 16.67 130
19 0.125 0.250 6 × 106 6 × 106 2.2 × 106 2.0 × 106 63.33 66.67 230
21a 0.125 0.250 6 × 106 6 × 106 2.2 × 106 2.0 × 106 63.33 66.67 90
21b 0.125 0.250 6 × 106 6 × 106 3.5 × 106 2.0 × 106 41.67 66.67 210
zanamivir 0.125 0.250 6 × 106 6 × 106 5.0 × 104 1.1 × 104 99.17 99.81  
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a

Results are expressed as mean ± standard deviation where multiple assays were performed.

b

Not applicable

Figure 4.

Figure 4

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Antiviral activity of the tested compounds using the plaque reduction assay.

Scheme 5. Synthesis of Acyclic Thioglycosides 21a,b and Their Conversion to Pyrazole 4.

Scheme 5

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Experimental Section

Chemistry

Melting points were measured with a Stuart melting point apparatus and were uncorrected. The IR spectra were measured using a PerkinElmer Spectrum 2 FTIR spectrophotometer using the ATR technique. The 1H NMR spectra were recorded using a Bruker NMR spectrometer at 400 MHz with DMSO-d6 as a solvent and TMS as an internal standard. Chemical shifts were reported as δ values in ppm. Mass spectra were recorded using a Shimadzu GCMS–QP 1000 EX mass spectrometer in the EI (70 eV) mode. Elemental analyses were carried out at the Microanalytical Center, Cairo University. Precoated silica gel 60.778 plates (Fluka) were used to perform analytical thin-layer chromatography, and the spots were recorded with a UV light at 254 nm. A potential cytotoxicity assay of the newly synthesized compounds was performed at the Center of Scientific Excellence for Influenza Viruses, Environmental Research Division, National Research Centre (NRC), Dokki, Cairo 12,622, Egypt.

General Procedure for the Synthesis of N-(2,2-Dicyano-1-(alkylthio)vinyl)benzamides 3a–c

Method: a mixture of malononitrile (0.66 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 (0.65 g, 0.01 mol). After cooling, methyl iodide (2.13 g, 0.015 mol), ethyl iodide (1.56 g, 0.015 mol), or benzyl chloride (1.26 g, 0.01 mol) was added. The reaction mixture was stirred overnight, and the resulting precipitate was filtered off, washed with water, dried, and recrystallized from ethanol.

N-(2,2-Dicyano-1-(methylthio)vinyl)benzamide 3a

Yellow powder; (ethanol); yield 85%; mp 254–256 °C; 1H NMR (400 MHz, DMSO-d6): δ 2.73 (s, 3H, CH3), 7.58–7.71 (m, 3 H, 3 CH-aromatic), 8.21–8.23 (d, 2H, aromatic), 13.53 (br s, H, NH); 13C NMR (100 MHz, DMSO-d6): δ 13.4, 92.54, 114.90, 128.84, 129.35, 131.38, 133.00, 158,56, 175.81; IR (cm–1): υ 2222 (CN), 1659 (C=O); EI-MS: m/z 243 [M+]; Anal.: Calcd for C12H9N3OS (243.3): C, 59.24; H, 3.73; N, 17.27. Found: C, 59.54; H, 4.03; N, 17.67.

N-(2,2-Dicyano-1-(ethylthio)vinyl)benzamide 3b

Orange powder; (ethanol); yield 75%; mp 250–255 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.36–1.40 (t, 3H, CH3), 3.32–3.37 (q, 2H, CH2), 7.61–8.21 (m, 5H, aromatic), 13.49 (br s, H, NH); IR(cm–1): υ 3681 (NH), 2214 (CN), 1654 (C=O); Anal.: Calcd for C13H11N3OS (257.3): C, 60.68; H, 4.31; N, 16.33. Found: C, 60.99; H, 4.91; N, 16.83.

N-(1-(Benzylthio)-2,2-dicyanovinyl)benzamide 3c

White powder; (ethanol); yield 63%; mp 250–260 °C; 1H NMR (400 MHz, DMSO-d6): δ 4.68 (s, 2H, CH2), 7.25–8.22 (m, 10H, aromatic), 13.47 (br s, H, NH); 13C NMR (100 MHz, DMSO-d6): 34.18, 114.82, 127.91, 129.08, 129.38, 129.43, 131.49, 133.83, 137.50, 158.86, 174.60; IR (cm–1): υ 3663 (NH), 2218 (CN), 1654 (C=O); Anal.: Calcd for C18H13N3OS (319.4): C, 67.69; H, 4.10; N, 13.16. Found: C, 67.97; H, 4.8; N, 13.66.

Synthesis of N-(5-Amino-4-cyano-1H-pyrazol-3-yl)benzamide 4

Method: hydrazine hydrate (0.50 g, 0.01 mol) was added to a solution of N-[2,2-dicyano-1-(alkylsulfanyl)ethenyl]benzamide (3a–c) (0.01 mol) in ethanol (20 mL) in the presence of drops of piperidine. The mixture was heated under reflux with continuous stirring for 3 h and then poured onto ice. The solid product was filtered off, dried, and recrystallized from ethanol to afford compound 4.

N-(5-Amino-4-cyano-1H-pyrazol-3-yl)benzamide 4

Yellow powder; (ethanol); yield 88%; mp 311 °C; 1H NMR (400 MHz, DMSO-d6): δ 5.37 (br s, 2H, NH2), 7.50–8.09 (m, 5H, aromatic), 11.73 (br s, H, NH), 12.33 (br s, H, NH); IR (cm-1): υ 3680 (NH2), 1639 (C=O); EI-MS: m/z = 227[M+]; Anal.: Calcd for C11H9N5O (227.2): C, 58.14; H, 3.99; N, 30.82. Found: C, 58.64; H, 3.69; N, 30.42.

Synthesis of N-(Pyrazolo[5,1-c][1,2,4]triazin-7-yl)benzamides 6a,b

Method: a solution of sodium nitrite (0.68 g, 0.01 mol) was gradually added to a cold solution of N-(5-amino-4-cyano-1H-pyrazol-3-yl) benzamide 4 (2.27 g, 0.01 mol) in hydrochloric acid (2 mL, 36%). The diazonium salt obtained was added to malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) in ethanol (10 mL) containing sodium acetate anhydrous (0.5 g) at 0 °C. The reaction mixture was stirred for 2 h, and the colored solid formed was filtered, washed with water, and crystallized from ethanol.

N-(4-Amino-3,8-dicyanopyrazolo[5,1-c][1,2,4]triazin-7-yl)benzamide 6a

Orange powder; (ethanol); yield 90%; mp>300 °C; 1H NMR (400 MHz, DMSO-d6): δ 3.60 (br s, 2H, NH2), 7.46–8.19 (m, 5H, aromatic), 10.47 (br s, H, NH); 13C NMR (100 MHz, DMSO-d6): δ 93.86, 115.98, 117.85, 128.43, 129.08, 132.14, 133.21, 144.09, 145.57, 157.49, 159.67, 172.71; Anal.: Calcd for C14H8N8O (304.3): C, 55.26; H, 2.65; N, 36.83. Found: C, 55.66; H, 2.95; N, 36.53.

N-(3,8-Dicyano-4-ethoxypyrazolo[5,1-c][1,2,4]triazin-7-yl)benzamide 6b

Yellow powder; (ethanol); yield 75%; mp>300 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.33–1.37 (t, 3H, CH3), 4.34–4.39 (q, 2H, CH2), 7.54–8.23 (m, 5H, aromatic), 12.52 (s, H, NH); Anal.: Calcd for C16H11N7O2 (333.3): C, 57.66; H, 3.33; N, 29.42. Found: C, 57.96; H, 3.83; N, 29.92.

Synthesis of N-(Cycloalka[e]pyrazolo[1,5-a]pyrimidin-2-yl)benzamides 8a,b

Method: a solution of compound 4 (2.27 g, 0.01 mol) and [(E)-(2-oxo-cyclopentylidene) methoxy] sodium 7a (1.34 g, 0.01 mol) or [(E)-(2-oxocyclohexylidene) methoxy] sodium 7b (1.48 g, 0.01 mol) was refluxed for 10 min in the presence of piperidine acetate (1 mL; prepared from 4.2 mL of glacial acetic acid, 10 mL of water, and 7.2 mL of piperidine). Acetic acid (1.5 mL) was added to the hot solution, and refluxing was continued for about 15 min. The reaction mixture was allowed to cool to room temperature. The precipitate, in each case, was collected by filtration and crystallized from ethanol.

N-(3-Cyano-7,8-dihydro-6H-cyclopenta[e]pyrazolo[1,5-a]pyrimidin-2-yl)benzamide 8a

Black powder; (ethanol); yield 60%; mp>300 °C; 1H NMR (400 MHz, DMSO-d6): δ 2.30–2.36 (m, 2H, CH2), 3.16–3.20 (t, 2H, CH2), 3.42–3.46 (t, 2H, CH2), 7.55–8.24 (m, 5H, aromatic), 8.80 (s, H, pyrimidine); Anal.: Calcd for C17H13N5O (303.3): C, 67.32; H, 4.32; N, 23.09. Found: C, 67.62; H, 4.82; N, 23.69.

N-(3-Cyano-6,7,8,9-tetrahydrocyclohexa[e]pyrazolo[1,5-a]pyrimidin-2-yl)benzamide 8b

Yellow solid; (ethanol); yield 75%; mp>300 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.81(br s, 2H, CH2), 1.98 (br s, 2H, CH2), 2.88 (br s, 2H, CH2), 3.15 (br s, 2 H, CH2), 7.54–8.20 (m, 5H, aromatic), 8.61 (s, H, pyrimidine), 12.12 (br s, H, NH); 13C NMR (100 MHz, DMSO-d6): δ 21.04, 21.56, 22.34, 24.51, 121.82, 128.46, 129.13, 132.19, 133.13, 145.91, 152.51, 157.27, 159.10; Anal.: Calcd for C18H15N5O (317.3): C, 68.13; H, 4.76; N, 22.07. Found: C, 68.63; H, 4.46; N, 22.77.

Synthesis of N-(5-amino-3,6-dicyano-7-arylpyrazolo[1,5-a]pyrimidin-2-yl)benzamides 10a–c

Method: a mixture of compound 4 (2.27 g, 0.01 mol) and benzylidene propanedinitrile 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; 1H NMR (400 MHz, DMSO-d6): δ 7.38–8.37 (m, 10H, aromatic), 8.71 (s, 2H, NH2), 12.22 (br s, H, NH); Anal.: Calcd for C21H13N7O (379.4): C, 66.48; H, 3.45; N, 25.84. Found: C, 66.78; H, 3.95; N, 25.54.

N-(5-Amino-3,6-dicyano-7-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-2-yl)benzamide 10b

Yellow powder; (DMF); yield 85%; mp>300 °C; 1H NMR (400 MHz, DMSO-d6): δ 3.88 (s, 3H, CH3), 7.15–8.23 (m, 9H, aromatic), 9.18(s, 2H, NH2), 12.19 (br s, H, NH); 13C NMR (100 MHz, DMSO-d6): δ 55.92, 91.75, 96.63, 114.28, 126.10, 128.51, 129.18, 131.12, 132.94, 150.99, 155.00, 157.94, 160.87, 177.35; IR (cm–1): υ 3439 (NH2), 2209 (CN), 1687 (C=O); EI-MS: m/z 409 [M+]; Anal.: Calcd for C22H15N7O2 (409.4): C, 64.54; H, 3.69; N, 23.95. Found: C, 64.94; H, 3.99; N, 23.65.

N-(5-Amino-7-(4-chlorophenyl)-3,6-dicyanopyrazolo[1,5-a]pyrimidin-2-yl)benzamide 10c

Yellow powder; (DMF); yield 55%; mp>300 °C; 1H NMR (400 MHz, DMSO-d6): δ 7.56–8.23 (m, 9H, aromatic), 9.25(br s, 2H, NH2), 12.08 (br s, H, NH); Anal.: Calcd for C21H12ClN7O (413.8): C, 60.95; H, 2.92; N, 23.69. Found: C, 60.65; H, 2.52; N, 23.99.

Synthesis of N-(3-Cyano-5-methylpyrazolo[1,5-a]pyrimidin-2-yl)benzamide 12a,b

Method: a mixture of compound 4 (2.27 g, 0.01 mol) and acetylacetone 11a (1.00 g, 0.01 mol) or ethyl acetoacetate 11b (1.30 g, 0.01 mol) was refluxed for 3 h in ethanol (25 mL) in the presence of drops of piperidine. The reaction mixture was allowed to cool to room temperature. The precipitate was collected by filtration and crystallized from ethanol.

N-(3-Cyano-5,7-dimethylpyrazolo[1,5-a]pyrimidin-2-yl)benzamide 12a

White powder; (ethanol); yield 75%; mp>300 °C; 1H NMR (400 MHz, DMSO-d6): δ 2.65 (s, 3H, CH3), 2.81 (s, 3H, CH3), 7.34 (s, H, CH-pyrimidin), 7.55–8.23 (m, 5H, aromatic), 12.21 (s, H, NH); 13C NMR (100 MHz, DMSO-d6): δ 17.48, 24.70, 112.82, 128.46, 129.13, 132.22, 133.10, 146.95, 157.38, 159.20, 161.61, 162.58; IR (cm–1): υ 3680 (NH), 1673 (C=O); EI-MS: m/z 291 [M+]; Anal.: Calcd for C16H13N5O (291.3): C, 65.97; H, 4.50; N, 24.04. Found: C, 65.67; H, 4.80; N, 24.74.

N-(3-Cyano-7-hydroxy-5-methylpyrazolo[1,5-a]pyrimidin-2-yl)benzamide 12b

Yellow powder; (ethanol); yield 55%; mp>300 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.91 (s, 1H, OH), 2.38 (s, 3H, CH3), 5.93 (s, H, CH-pyrimidin), 7.54–8.17 (m, 5H, aromatic), 11.99 (s, H, NH); Anal.: Calcd for C15H11N5O2 (293.3): C, 61.43; H, 3.78; N, 23.88. Found: C, 61.93; H, 3.48; N, 23.58.

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.

(E)-Ethyl 3-benzamido-2-cyano-3-(methylthio)acrylate 14

Yellow powder; (ethanol); yield 53%; mp 64 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.36–1.41 (t, 3H, CH3), 2.39 (s, 3H, S-CH3) , 4.52–4.57 (q, 2H, CH2), 7.49–8.07 (m, 5H, aromatic), 8.05–8.07 (s, H, NH); Anal.: Calcd for C14H14N7O3S (290.3): C, 57.92; H, 4.86; N, 9.65. Found: C, 57.62; H, 4.56; N, 9.95.

Synthesis of 3-Ethoxy-5-phenyl-1H-1,2,4-triazole 19

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, and recrystallized from ethanol to afford compound 19.

3-Ethoxy-5-phenyl-1H-1,2,4-triazole 19

Colorless crystal; (ethanol); yield 60%; mp 118–120 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.37 (br s, 3H, CH3), 4.34 (br s, 2H, CH2) , 7.47–7.93 (S, 5H, aromatic), 13.72 (s, H, NH); IR (cm–1): υ 3680 (NH); Anal.: Calcd for C10H11N3O (189.21): C, 63.48; H, 5.86; N, 22.21. Found: C, 63.25; H, 5.62; N, 22.44.

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 (25 mL) in the presence of potassium hydroxide (0.56 g, 0.01 mol). After cooling, a solution of 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide 20 (4.11 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 thin-layer chromatography using DCM as an eluent where compounds 21a,b showed a value of R.F. equals to 0.85–0.87.

(2R,3S,4R,5R,6S)-2-(Acetoxymethyl)-6-((1-benzamido-2,2-dicyanovinyl)thio)tetrahydro-2H-pyran-3,4,5-triyl triacetate 21a

Yellow powder; yield 75%; mp>300 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.95–2.01 (4 s, 12H, 4x OAc), 3.93–4.01 (m, 2H, 2H-6′), 4.31 (t, H, H-4′), 4.91–4.97(t, H, H-5′), 5.10–5.16 (t, H, H-3′), 5.64–5.69 (t, H, H-2′), 6.17–6.21 (d, H, H-1′, J1′,2′ = 14.4), 7.21–8.34 (m, 5H, aromatic); Anal.: Calcd for C25H25N3O10S (559.5): C, 53.66; H, 4.50; N, 7.51. Found: C, 55.96; H, 4.90; N, 7.81.

(2R,3S,4R,5R,6S)-2-(Acetoxymethyl)-6-(((E)-1-benzamido-2-cyano-3-ethoxy-3-oxoprop-1-en-1-yl)thio)tetrahydro-2H-pyran-3,4,5-triyl triacetate 21b

Yellow powder; yield 84%; mp>300 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.18–1.27 (t, 3H, CH2-CH3), 1.59–2.00 (4 s, 12H, 4x OAc), 3.92–4.04 (m, 3H, 2H-6′ & H-4′), 4.22–4.27(q, 2H, CH2-CH3), 4.89–4.94 (t, H, H-5′), 5.03–5.08 (t, H, H-3′), 5.61–5.64(t, H, H-2′), 6.10–6.12 (d, H, H-1′, J1′,2′ = 9.2), 7.56–8.32 (m, 5H, aromatic), 13.20 (s, H, NH); 13C NMR (100 MHz, DMSO-d6): δ 14.50, 19.01, 20.50, 20.86, 20.94, 56.43, 61.50, 68.56, 69.47, 73.47, 75.10, 80.08, 92.51, 121.11, 123.06, 129.31, 133.89, 137.23, 164.86, 166.53, 169.77, 170.03, 170.43; IR (cm–1): υ 3697 (NH), 2193 (CN), 1744 (C=O); Anal.: Calcd for C27H30N2O12S (606.6): C, 53.46; H, 4.98; N, 4.62. Found: C, 53.76; H, 4.58; N, 4.92.

Antiviral Activity

MTT Cytotoxicity Assay (LD50)

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)-2,5-diphenyltetrazolium bromide)51 with some minor modifications. Cells were placed in 96-well plates and incubated for 1 day at room temperature in 5% CO2. 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 λmax 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.

graphic file with name ao0c02675_m001.jpg

The plot of cytotoxicity percentage versus concentration of sample was used to calculate the concentration, which showed 50% cytotoxicity (LD50).

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 H2O. 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:

graphic file with name ao0c02675_m002.jpg

Acknowledgments

We thank Prof. Dr. Mohamed Ahmed Ali, Director of Center of Scientific Excellence for Influenza Viruses (CSEIV), the National Research Center, for performing biological assay tests.

Supporting Information Available

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, 1H NMR, and 13C NMR spectra for the newly synthesized compounds (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao0c02675_si_001.pdf (1.6MB, pdf)

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