Synthesis, anticancer evaluation, and molecular modeling study of new 2-(phenylamino)pyrazolo[1,5-a]pyrimidine analogues

Omer A. Azher; Aisha Hossan; Rami A. Pashameah; Amerah Alsoliemy; Arwa Alharbi; Turki M. Habeebullah; Nashwa M. El-Metwaly

doi:10.1016/j.arabjc.2022.104437

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Original article

01 2022

:16;

104437

doi:

10.1016/j.arabjc.2022.104437

Synthesis, anticancer evaluation, and molecular modeling study of new 2-(phenylamino)pyrazolo[1,5-a]pyrimidine analogues

Omer A. Azher^{^a}, Aisha Hossan^{^b}, Rami A. Pashameah^{^c}, Amerah Alsoliemy^{^c}, Arwa Alharbi^{^c}, Turki M. Habeebullah^{^d}, Nashwa M. El-Metwaly^{^c,^e,⁎}

a Department of laboratory Medicine, Faculty of Applied Biomedical Sciences, Al-Baha University, Saudi Arabia

b Department of Chemistry, Faculty of science, King Khalid University, Abha, Saudi Arabia

c Department of Chemistry, Faculty of Applied Science, Umm Al Qura University, Makkah 24230, Saudi Arabia

d Department of Environment and Health Research, The Custodian of the two holy mosques Institute for Hajj and Umrah Research, Umm Al Qura University, Makkah, Saudi Arabia

e Department of Chemistry, Faculty of Science, Mansoura University, El-Gomhoria Street 35516, Egypt

⁎Corresponding author. N_elmetwaly00@yahoo.comnmm (Nashwa M. El-Metwaly) nmmohamed@uqu.edu.sa (Nashwa M. El-Metwaly)

Received: 2022-8-18, Accepted: 2022-11-13,

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

The reaction of 3-amino-5-phenylaminopyrazoles 2 with 3-(dimethylamino) acrylonitrile derivatives resulted in a series of substituted pyrazolopyrimidine analogues 4 and 6. The DFT studies of the isolated compounds showed that the frontier molecular orbitals energy gap was close and in the 2.65–2.81 eV range where the derivative 6b has the lowest and both of 4a and 4c have the highest values. Meanwhile, the anticancer activity of the newly synthesized pyrazolopyrimidine analogues have been tested against several different cell lines (MCF-7, PC3, Hep-2 and WI38). The investigated pyrazolopyrimidines showed remarkable cytotoxicity activity against the MCF-7 and Hep-2 cell lines. In comparison to the effects of 5-fluorouracil, IC₅₀ = 10.19 ± 0.42 and 7.19 ± 0.47, compounds 6a-c demonstrated potential anticancer activity with IC₅₀ values for MCF-7 (10.80 ± 0.36–19.84 ± 0.49 μM) and Hep-2 (8.85 ± 0.24–12.76 ± 0.16 μM). Important details regarding the protein's binding sites were disclosed when the produced analogues docked with the crystal structure of the KDM5A protein, which was located in the protein data library.

Keywords

3-aminopyrazoles

Pyrazolopyrimidines

Molecular modelling

MCF-7

Molecular docking

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1 Introduction

A significant heterocyclic moiety, the pyrimidine moiety exhibits a wide range of biological and pharmacological activity (Sathisha, Gopala et al. 2016, Jubeen, Iqbal et al. 2018, Nerkar 2021 ). Nucleotides, nucleic acids, vitamins, coenzymes, purines, pterins, and uric acids are examples of naturally occurring molecules that contain this six-membered 1,3-diazine ring with nitrogen at the 1 and 3 positions (Johnson and Khan 2018). The numerous medicinal uses of pyrimidine may be explained by the fact that it is a component of DNA and RNA (Joshi, Nayyar et al. 2016, Taylor, Houlihan et al. 2019). The first analogues to be examined for biological action were 5-halogenated analogues of pyrimidine moiety (Alcolea Palafox 2014, Matyugina, Logashenko et al. 2015). This heterocyclic moiety is part of several drugs like a fused, stiff, and planar N-heterocyclic system that contains both of pyrazole and pyrimidine moieties is known as the pyrazolo[1,5-a]pyrimidine (PP) (Arias-Gómez, Godoy et al. 2021, Mahapatra, Prasad et al. 2021). Due to its exceptional synthetic flexibility, this fused pyrazole is a preferred scaffold for combinatorial library design and drug discovery (Mackman, Sangi et al. 2015, Shirvani, Fassihi et al. 2019). This elasticity enables structural modifications all the way around its border. After the chief critical evaluation involving this interesting scaffold (Jismy, Tikad et al. 2020), several reviews regarding to the achievement and subsequent derivatization procedures have been designated in the literature (El Sayed, Hussein et al. 2018, Pinheiro et al. 2020, Kumar, Das et al. 2021 ). In spite of these discoveries, the synthetic alterations covering this theme continue to be an exploration in terms of the effectiveness of the development, the influence on the setting, and the investigation of its various uses. The methods that aim to shorten synthesis routes, use affordable reagents, and create waste-prevention or -reduction techniques should be covered in these studies. Typically, the preparation of PP analogues includes the formation of pyrimidine rings by reaction with various 1,3-biselectrophilic reagents with aminopyrazoles (Castillo and Portilla 2018, Al-Azmi 2019, Salem, Helal et al. 2019 ). Pyrazolo[1,5-a]pyrimidine analogues is considered an important fragment for bioactive materials through their unique properties corresponding to selectivity as a protein inhibitor (Asano, Yamazaki et al. 2012), anticancer (Zhao, Ren et al. 2016), between others outstanding qualities (Lunagariya, Thakor et al. 2018, Ali, Ibrahim et al. 2019, Almehmadi, Alsaedi et al. 2021 ). Additionally, the pyrazolo[1,5-a]pyrimidine biocompatibility and lower toxicity resulted in a wide range derivatives to be employed as bioactive materials under commercial names like “Zaleplon, Indiplon, Lorediplon, Reversan, Dorsomorphin, Pyrazophos, Anagliptin, and Presatovir” (Fig. 1) (Eftekhari-Sis and Zirak 2015). Recently, this molecular component has been the subject of research for potential new uses in the field of materials sciences, because of its outstanding photophysical characteristics as an emerging fluorophore (Castillo, Rosero et al. 2017, Tigreros, Aranzazu et al. 2020, Tigreros, Macias et al. 2021, Tigreros, Zapata-Rivera et al. 2021 ). Similarly, the propensity of pyrazolo[1,5-a]pyrimidine analogues to take the crystalline shapes with prominent conformations and supramolecular behaviors (Secrieru, O’Neill et al. 2019, Tigreros, Macías et al. 2022) could magnify their uses against the solid-state. From the previous literatures as substituted PPs presented good bioactivity, it encouraged us to synthesis another PP substituted with different groups such as substituted amide, substituted amino, and nitrile groups besides study their anticancer activities toward different cell lines in addition to study their theoretical activity through modeling and docking stimulation toward cancer protein.

Chemical structures of some derivatives bearing pyrazolopyrimidine motif.

2 Experimental

2.1

2.1 General remarks

Melting point measurements are performed using the Gallenkamp electric apparatus. The IR spectra were captured using a Thermo Scientific Nicolet iS10 FTIR spectrometer (KBr discs). The NMR spectra were obtained in DMSO‑d₆ using a JEOL NMR spectrometer at 500 MHz (¹H NMR) and 125 MHz (¹³C NMR). The mass analyses were carried out on a Thermo Scientific Focus/DSQII Quadrupole GC/MS (70 eV). The C, H, and N elemental analyses were carried out using a Perkin-Elmer 2400 analyzer.

2.2

2.2 Preparation of 3-amino-N-aryl-5-(phenylamino)-1H-pyrazole-4-carboxamide analogues 2a-c

Hydrazine hydrate 80 % (1.00 ml, 20 mmol) was added to each solution of the ketene S,N-acetal derivative 1a, 1b, or 1c (15 mmol) in 40 ml of ethanol. The mixture in each case was heated under reflux for 6 h and the precipitated solid was filtered to pick up the targeting 3-amino-5-phenylamino-pyrazole derivatives 2a, 2b, and 2c, respectively (Elgemeie, Elghandour et al. 2004, Elgemeie and Jones 2004, Mukhtar, Hassan et al. 2021 ).

2.3

2.3 Preparation of 7-amino-N-aryl-6-cyano-2-(phenylamino)-pyrazolo[1,5-a]pyrimidine-3-carboxamide analogues 4a-c

To a solution of 3-amino-5-phenylamino-pyrazole derivative 2a, 2b, or 2c (5 mmol) in 40 ml dioxane and 0.2 ml triethylamine, 2-((dimethylamino)methylene)-malononitrile (3) (0.60 g, 5 mmol) was added. The solution was heated under reflux for 3–4 h, the progress of the reaction was monitored by TLC. After completion of the reaction, the mixture was cooled to 30 °C. Crystallization of the obtained solid from a dioxane yielded the conforming pyrazolo[1,5-a]pyrimidine analogues 4a, 4b, and 4c, respectively.

7-Amino-6-cyano-2-(phenylamino)-N-(p-tolyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (4a):

Yellow crystals, yield = 71 %, m.p. = 222–223 °C, R_f = 0.43 (hexane:EtOAc = 3:1). IR (ν/cm⁻¹): 3343, 3305, 3267, and 3190 (NH₂ and 2 N—H), 3056 (C—H, aromatic), 2941 (C—H, aliphatic), 2210 (C≡N), 1661 (C⚌O). ¹H NMR (δ/ppm): 2.29 (s, 3H, –CH₃), 6.87 (s, 2H, exchangeable by D₂O, NH₂), 7.14 (t, J = 7.50 Hz, 1H), 7.31 (d, J = 8.50 Hz, 2H), 7.40 (t, J = 8.50 Hz, 2H), 7.46 (d, J = 8.50 Hz, 2H), 7.58 (d, J = 8.50 Hz, 2H), 8.58 (s, 1H, pyrimidine-H), 10.25 (s, 1H, exchangeable by D₂O, N—H), 11.68 (s, 1H, exchangeable by D₂O, N—H). ¹³C NMR (δ/ppm): 21.31 (–CH₃), 91.08 (C-6), 98.14 (C-3), 116.82 (C≡N), 118.64 (2 Ph-C), 121.11 (2 Ar-C), 123.54 (Ph-C), 129.35 (2 Ar-C), 129.98 (2 Ph-C), 134.67 (Ar-C), 136.70 (Ar-C), 139.96 (Ph-C), 151.38 (fused-C), 155.19 (C-2), 160.47 (C-5), 162.86 (C⚌O), 166.23 (C-7). MS m/z (%): 383 (M⁺, 27.80). Analysis for C₂₁H₁₇N₇O (383.15): Calcd.: C, 65.79; H, 4.47; N, 25.57 %. Found: C, 65.91; H, 4.41; N, 25.65 %.

7-Amino- N-(p-anisyl)-6-cyano-2-(phenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (4b):

Yellow crystals, yield = 65 %, m.p. = 230–231 °C, R_f = 0.54 (hexane:EtOAc = 3:1). IR (ν/cm⁻¹): 3332, 3294, 3245, and 3192 (NH₂ and 2 N—H), 3067 (C—H, aromatic), 2951 (C—H, aliphatic), 2211 (C≡N), 1658 (C⚌O). ¹H NMR (δ/ppm): 3.79 (s, 3H, –OCH₃), 6.91 (s, 2H, exchangeable by D₂O, NH₂), 7.04 (d, J = 9.00 Hz, 2H), 7.19 (t, J = 7.50 Hz, 1H), 7.46 (t, J = 7.50 Hz, 2H), 7.56 (d, J = 9.00 Hz, 2H), 7.75 (d, J = 9.00 Hz, 2H), 8.62 (s, 1H, pyrimidine-H), 10.43 (s, 1H, exchangeable by D₂O, N—H), 11.56 (s, 1H, exchangeable by D₂O, N—H). ¹³C NMR (δ/ppm): 56.04 (–OCH₃), 90.87 (C-6), 97.44 (C-3), 114.91 (2 Ar-C), 116.57 (C≡N), 118.69 (2 Ph-C), 122.42 (2 Ar-C), 123.58 (Ph-C), 130.04 (2 Ph-C), 132.63 (Ar-C), 140.18 (Ph-C), 151.75 (fused-C), 154.80 (C-2), 159.05 (Ar-C), 161.17 (C-5), 163.24 (C⚌O), 165.89 (C-7). MS m/z (%): 399 (M⁺, 43.29). Analysis for C₂₁H₁₇N₇O₂ (399.14): Calcd.: C, 63.15; H, 4.29; N, 24.55 %. Found: C, 63.01; H, 4.22; N, 24.44 %.

7-Amino-N-(p-chlorophenyl)-6-cyano-2-(phenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (4c):

Yellowish brown crystals, yield = 68 %, m.p. = 243–244 °C, R_f = 0.46 (hexane:EtOAc = 3:2). IR (ν/cm⁻¹): 3343, 3276, 3202 (NH₂ and 2 N—H), 3083 (C—H, aromatic), 2214 (C≡N), 1665 (C⚌O). ¹H NMR (δ/ppm): 6.94 (s, 2H, exchangeable by D₂O, NH₂), 7.13 (t, J = 7.50 Hz, 1H), 7.34 (t, J = 7.50 Hz, 2H), 7.48 (d, J = 9.00 Hz, 2H), 7.56 (d, J = 9.00 Hz, 2H), 7.70 (d, J = 7.50 Hz, 2H), 8.60 (s, 1H, pyrimidine-H), 10.41 (s, 1H, exchangeable by D₂O, N—H), 10.94 (s, 1H, exchangeable by D₂O, N—H). ¹³C NMR (δ/ppm): 90.38 (C-6), 98.87 (C-3), 116.43 (C≡N), 118.55 (2 Ph-C), 121.69 (2 Ar-C), 123.30 (Ph-C), 128.85 (2 Ar-C), 129.81 (2 Ph-C), 133.17 (Ar-C), 135.93 (Ar-C), 139.49 (Ph-C), 152.07 (fused-C), 155.66 (C-2), 161.89 (C-5), 162.52 (C⚌O), 166.14 (C-7). MS m/z (%): 403 (M⁺, 36.54). Analysis for C₂₀H₁₄ClN₇O (403.09): Calcd.: C, 59.49; H, 3.49; N, 24.28 %. Found: C, 59.67; H, 3.44; N, 24.15 %.

2.4

2.4 Synthesis of 7-amino-N₃-aryl-2-(phenylamino)-pyrazolo[1,5-a]pyrimidine-3,6-dicarboxamide analogues 6a-c

A mixture of each 3-aminopyrazole compound 2a, 2b, or 2c (5 mmol) 2-cyano-3-(dimethylamino)acrylamide (5) (0.69 g, 5 mmol) in 30 ml dioxane and 0.2 ml triethylamine was heated under reflux for 3–4 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the solid that obtained upon cooling to 30 °C was filtered and crystallized EtOH/DMF mixture (2:1) to give the corresponding pyrazolo[1,5-a]pyrimidine analogues 6a, 6b, and 6c, respectively.

7-Amino-2-(phenylamino)–N₃-(p-tolyl)pyrazolo[1,5-a]pyrimidine-3,6-dicarboxamide (6a):

Orange crystals, yield = 62 %, m.p. = 265–266 °C, R_f = 0.33 (hexane:EtOAc = 1:1). IR (ν/cm⁻¹): 3369, 3311, 3260, 3194 (NH₂ and 2 N—H), 3062 (C—H, aromatic), 2938 (C—H, aliphatic), 1658 (C⚌O). ¹H NMR (δ/ppm): 2.32 (s, 3H, –CH₃), 6.70 (s, 2H, exchangeable by D₂O, NH₂), 7.07 (t, J = 7.50 Hz, 1H), 7.28–7.35 (m, 4H), 7.44 (d, J = 8.50 Hz, 2H), 7.57 (d, J = 8.50 Hz, 2H), 7.88 (s, 2H, exchangeable by D₂O, NH₂), 8.18 (s, 1H, pyrimidine-H), 10.52 (s, 1H, exchangeable by D₂O, N—H), 11.37 (s, 1H, exchangeable by D₂O, N—H). ¹³C NMR (δ/ppm): 21.33 (–CH₃), 98.51 (C-3), 104.29 (C-6), 118.78 (2 Ph-C), 120.93 (2 Ar-C), 123.47 (Ph-C), 129.27 (2 Ar-C), 129.97 (2 Ph-C), 135.48 (Ar-C), 136.17 (Ar-C), 140.36 (Ph-C), 143.62 (fused-C), 155.39 (C-2), 158.96 (C-5), 163.21 (C⚌O), 165.56 (C-7), 167.09 (C⚌O). MS m/z (%): 401 (M⁺, 67.11). Analysis for C₂₁H₁₉N₇O₂ (401.16): Calcd.: C, 62.83; H, 4.77; N, 24.42 %. Found: C, 62.94; H, 4.69; N, 24.50 %.

7-Amino-N₃–(p-anisyl)-2-(phenylamino)pyrazolo[1,5-a]pyrimidine-3,6-dicarboxamide (6b):

Brown powder, yield = 60 %, m.p. = 271–272 °C, R_f = 0.41 (hexane:EtOAc = 1:2). IR (ν/cm⁻¹): 3337, 3282, 3229 (NH₂ and 2 N—H), 3078 (C—H, aromatic), 2954 (C—H, aliphatic), 1655 (C⚌O). ¹H NMR (δ/ppm): 3.81 (s, 3H, –OCH₃), 7.01 (d, J = 9.00 Hz, 2H), 7.07 (s, 2H, exchangeable by D₂O, NH₂), 7.17 (t, J = 7.50 Hz, 1H), 7.38 (d, J = 7.50 Hz, 2H), 7.56 (t, J = 7.50 Hz, 2H), 7.72 (d, J = 9.00 Hz, 2H), 7.88 (s, 2H, exchangeable by D₂O, NH₂), 8.21 (s, 1H, pyrimidine-H), 10.26 (s, 1H, exchangeable by D₂O, N—H), 11.11 (s, 1H, exchangeable by D₂O, N—H). ¹³C NMR (δ/ppm): 56.05 (–OCH₃), 97.85 (C-3), 103.02 (C-6), 114.56 (2 Ar-C), 119.07 (2 Ph-C), 122.44 (2 Ar-C), 123.54 (Ph-C), 129.81 (2 Ph-C), 130.69 (Ar-C), 139.86 (Ph-C), 143.73 (fused-C), 154.98 (C-2), 158.46 (Ar-C), 159.13 (C-5), 163.05 (C⚌O), 164.82 (C-7), 166.95 (C⚌O). MS m/z (%): 417 (M⁺, 55.08). Analysis for C₂₁H₁₉N₇O₃ (417.15): Calcd.: C, 60.42; H, 4.59; N, 23.49 %. Found: C, 60.21; H, 4.48; N, 23.63 %.

7-Amino-N₃–(p-chlorophenyl)-2-(phenylamino)pyrazolo[1,5-a]pyrimidine-3,6-dicarboxamide (6c):

Brown powder, yield = 58 %, m.p. = 293–294 °C, R_f = 0.45 (hexane:EtOAc = 1:2). IR (ν/cm⁻¹): 3351, 3307, 3266, 3208 (NH₂ and 2 N—H), 3087 (C—H, aromatic), 1657 (C⚌O). ¹H NMR (δ/ppm): 6.79 (s, 2H, exchangeable by D₂O, NH₂), 7.09 (t, J = 7.50 Hz, 1H), 7.28 (d, J = 8.50 Hz, 2H), 7.33–7.38 (m, 4H), 7.63 (d, J = 8.50 Hz, 2H), 7.67 (s, 2H, exchangeable by D₂O, NH₂), 8.24 (s, 1H, pyrimidine-H), 10.58 (s, 1H, exchangeable by D₂O, N—H), 11.46 (s, 1H, exchangeable by D₂O, N—H). ¹³C NMR (δ/ppm): 98.77 (C-3), 105.43 (C-6), 118.81 (2 Ph-C), 121.89 (2 Ar-C), 123.63 (Ph-C), 129.18 (2 Ar-C), 129.78 (2 Ph-C), 133.05 (Ar-C), 135.92 (Ar-C), 140.43 (Ph-C), 144.07 (fused-C), 156.70 (C-2), 158.56 (C-5), 162.91 (C⚌O), 165.12 (C-7), 167.25 (C⚌O). MS m/z (%): 423 (M⁺ + 2, 16.94), 421 (M⁺, 48.32). Analysis for C₂₀H₁₆ClN₇O₂ (421.11): Calcd.: C, 56.95; H, 3.82; N, 23.24 %. Found: C, 57.13; H, 3.89; N, 23.36 %.

2.5

2.5 Computational studies

The produced compounds were geometrically optimized using Gaussian 09 W program (Frisch, Trucks et al. 2009) at DFT/B3LYP level and 6–311⁺⁺G basis set (Lee, Yang et al. 1988, Perdew and Wang 1992, Becke 1993 ). Positive frequencies obtained for all derivatives were taken as evidence for the optimized geometries stability. The Materials Studio package DMol3 module (BIOVIA 2017) was employed for estimating the Fukui indices by applying the GGA and B3LYP functional methods with DNP basis set (version 3.5) (Delley 2006).

2.6

2.6 MTT assay

The produced pyrazolo[1,5-a]pyrimidine derivatives were tested for their ability to suppress the growth of the subsequent cancer cell lines: liver carcinoma (HepG2), laryngeal carcinoma (Hep-2), breast cancer (MCF-7), and prostate cancer PC3. Normal fibroblast cells were also used in the MTT test (WI38). In this test, a colorimetric method is used to change the yellow hue of “tetrazolium bromide” (MTT) into a purple formazan by utilizing mitochondrial succinate dehydrogenase. Cell lines were bred in the meantime using RPMI-1640 media with 10 % bovine serum at 37 °C with 5 % CO₂. Next incubation for 24 hrs., cells were uncovered to various concentrations of the produced hybrids. After this, the cells were brooded for a further 4 h before receiving 5 mg/ml of MTT thaw, Liquefaction of the produced purple formazan.

2.7

2.7 Molecular docking study

The estimation of molecular docking was carried out using the molecular docking application (MOE 2015.10). We obtained the PDB for the KDM5A structure from (https://www.rcsb.org/PDB codes- 5IVE). The following instructions were followed for arranging the produced protein. i) The structure of the enzyme is cleaned up of objective substances. ii) The MOE method allowed hydrogen atoms to be added to protein while minimizing process, with RMS values of 0.01 kcal.mol⁻¹ and an RMS range of 0.1. iii) The ligands were produced using the MOE builder interface.

3 Results and discussion

3.1

3.1 Preparation of 2-(phenylamino)pyrazolo[1,5-a]pyrimidine analogues 4 and 6

In light of the previous literature (Elgemeie, Elghandour et al. 2004, Elgemeie and Jones 2004, Mukhtar, Hassan et al. 2021 ), the building block 3-aminopyrazole derivatives 2a-c were prepared by reacting the ketene S,N-acetal compounds 1a-c with hydrazine hydrate in boiling ethanol for 6 h (Scheme 1).

Synthesis of 3-aminopyrazole scaffolds 2a-c.

The aminopyrazole compounds 2a-c are used as starting materials for the synthesis of various substituted pyrazolo[1,5-a]pyrimidine analogues. They reacted with 2-((dimethylamino)methylene)-malononitrile (3) to form the 7-amino-N-aryl-6-cyano-2-(phenylamino)-pyrazolo[1,5-a]pyrimidine-3-carboxamide analogues 4a-c (Scheme 2). The reaction was carried out by refluxing the reactants for 4 h in dioxane and triethylamine. The intermediate (A) is thought to be formed by the nucleophilic addition of an amino group (from pyrazole) to the β-carbon of acrylonitrile derivative 3. This intermediate (A) undergoes dimethylamine molecule elimination, and the resulting intermediate (B) cyclizes to the pyrimidine ring system via intramolecular addition of an internal N—H group (pyrazole) on the nitrile function. The structures of 7-aminopyrazolo[1,5-a]pyrimidine compounds 4a-c were established by their compatible spectral data. The IR spectrum of 4a (as an example) showed absorptions at 3343, 3305, 3267, and 3190 cm⁻¹ referring to the NH₂ and 2 N—H stretching frequencies. The absorptions that observed 2210 and 1661 cm⁻¹ were attributed to the nitrile (-C≡N), and amidic carbonyl (C⚌O) functions. ¹H NMR spectrum revealed singlet signals at δ 2.29 and 6.87 ppm for the protons of methyl and amino functions, respectively. The aromatic protons are resonated as triplet and doublet signals at in the region δ 7.14–7.58 ppm. The signal signals at δ 8.58, 10.25 and 11.68 ppm are attributed to the protons of pyrimidine-C and two N—H groups, respectively. The ¹³C NMR spectrum revealed seventeen signals for twenty-one carbon atoms. The characteristic signals of pyrimidine-CH and amide carbonyl are observed at δ 160.47 and 162.86 ppm. The mass analysis showed the molecular ion peak at m/z = 383 for the formula C₂₁H₁₇N₇O.

Synthesis of 2-(phenylamino)pyrazolo[1,5-a]pyrimidine analogues 4a-c.

The chemical reactivity of 3-aminopyrazoles 2a-c with 2-cyano-3-(dimethylamino)acrylamide (5) was also studied. To obtain the targeting 2-(phenylamino)pyrazolo[1,5-a]pyrimidine-3,6-dicarboxamide compounds 6a-c, the reaction was carried out in boiling dioxane and triethylamine (Scheme 3). The designed structures of compounds 6a-c were confirmed by their compatible spectral data. The IR spectrum of 6a showed absorptions at 3369, 3311, 3260, and 3194 cm¹ to indicate the stretching vibrations of NH₂ and two N—H groups. The amide carbonyl group is identified by the broad absorption near 1658 cm⁻¹. The ¹H NMR spectrum revealed singlet signals at δ 2.32 and 6.70 ppm for the protons of methyl and amino groups, respectively. The aromatic protons are observed as triplet, multiplet, and doublet signals at δ 7.07–7.57 ppm. The protons of amino group (CONH₂) are resonated as singlet at δ 7.88 ppm. The singlet signals that are resonated at δ 8.18, 10.52, and 11.37 ppm identified the pyrimidine-H and two N—H functions. The mass analysis showed the molecular ion peak at m/z = 401 for the formula C₂₁H₁₉N₇O₂.

Synthesis of 2-(phenylamino)pyrazolo[1,5-a]pyrimidine analogues 6a-c.

3.2

3.2 DFT molecular modeling study

The studied compounds optimized structures revealed that the 2-phenylamino pyrazolopyrimidine moiety have almost planar configuration where the nitrogen atom of the phenylamino groups were slightly shifted out plane, NH_(Pham)–C²_(Pp)-N¹_(Pp)-N⁸_(Pp) = 178.0° and N¹_(Pp)–C²_(Pp)–NH_(Pham)-C¹_(Pham) = 2.6°. Likewise, the amino and cyano substituents, in 4a-c derivatives, were coplanar with the pyrazolopyrimidine, e.g., NH_2(Pp)-C⁷_(Pp)-C⁶_(Pp)-C⁵_(Pp) = -179.4°, C⁵_(Pp)-C⁶_(Pp)–CN_(Pp)–NC_(Pp) = 179.3° and NH_2(Pp)-C⁷_(Pp)-C⁶_(Pp)–CN_(Pp) = 0.4°. Although, the carboxamide carbonyl carbon atoms were out the pyrazolopyrimidine plane, e.g., N⁴_(Pp)-C^3a_(Pp)–C³_(Pp)–CO_(carb) = 3.3°, whereas its oxygen and nitrogen atoms were strongly shifted out, i.e., C²_(Pp)–C³_(Pp)–CO_(carb)–OC_(carb) and C²_(Pp)–C³_(Pp)–CO_(carb)–NH_(carb) were −142.0° and 36.2°, respectively. In 6a-c derivatives, the carboxamide group, at position 6, were slightly shifted out the pyrazolopyrimidine plane, e.g., N⁸_(Pp)-C⁷_(Pp)-C⁶_(Pp)–CO_(carb1) = -179.1°, C⁷_(Pp)-C⁶_(Pp)–CO_(carb1)–OC_(carb1) = -2.0° and C⁵_(Pp)-C⁶_(Pp)–CO_(carb1)–NH_2(carb1) = -1.9°. (Fig. 2) (Table S1).

DFT Optimized structures of the 4a and 6a derivatives.

Additionally, the DFT calculated bond length and angle were almost match with those measured in comparable compounds single crystal X-ray (Liu and Liao 2006, Xiaobao, Li et al. 2008, Burnett, Johnston et al. 2015 ), i.e., lengths exhibited 0.00–0.13 Å difference and 0.048–0.059 Å RMSD while the angles were different by 0.0–11.5° with 2.7–5.2° RMSD, which were attributed to the absence intermolecular columbic interactions in the quantum chemical calculations, but the practical gained for molecules in solid crystal lattice (Sajan, Joseph et al. 2011) (Tables S2-S3).

3.2.1

3.2.1 Frontier molecular orbitals

The HOMO-LUMO configuration and energy explain the electron donating or receiving ability of the molecule (Bulat, Chamorro et al. 2004) where lesser energy gap leads to more ease intramolecular charge transfer (Xavier, Periandy et al. 2015, Makhlouf, Radwan et al. 2018) that may affect in the molecule’s biological activity (Bouchoucha, Zaater et al. 2018). The FMO 3D graph showed that the HOMO of all derivatives was localized mainly on the phenyl rings as well as the heteroatoms lone pair of electrons whilst the LUMO was principally constructed from the π*-orbitals of the 6-cyano and 6-carboxamide 3-carboxamide pyrazolopyrimidine moiety, in 4a-c and 6a-c, respectively (Fig. 3).(See Fig. 4).

The frontier molecular orbital of the investigated compounds.

IC50 of the cytotoxic activity for the examined derivatives against human tumor cell lines.

The abovementioned facts affected in the HOMO-LUMO energies, E_H and E_L. So, the data indicated that the E_H and E_L of all derivatives have close values of the, range −5.65 - −6.18 and −3.06 - −3.37 eV, respectively, where 4c has highest E_H and E_L. In addition, the derivatives 6b and both of 4a and 4c exhibited the minimum and maximum ΔE_H-L gap, 2.59–2.81 eV, the compounds may be ordered according to their energy gap as 4a = 4c > 6c ≈ 6a > 4b > 6b (Table 1).

Table 1 The HOMO-LUMO energies and chemical reactivity descriptors (eV) of investigated compounds.

Compound	E_H	E_L	ΔE_H-L	χ	η	δ	ω	Dipole
4a	−6.04	−3.24	2.81	4.64	1.40	0.71	7.67	6.27
4b	−5.82	−3.20	2.62	4.51	1.31	0.76	7.76	5.49
4c	−6.18	−3.37	2.81	4.77	1.40	0.71	8.11	6.17
6a	−5.83	−3.10	2.73	4.46	1.36	0.73	7.30	1.77
6b	−5.65	−3.06	2.59	4.36	1.30	0.77	7.32	0.86
6c	−5.96	−3.22	2.74	4.59	1.37	0.73	7.68	3.93

Furthermore, chemical reactivity descriptors, namely, electronegativity (χ), global hardness (η), softness (δ) and electrophilicity (ω) were determined utilizing the values of the E_H and E_L using the subsequent expressions (Xavier, Periandy et al. 2015). $χ = - \frac{1}{2} (E_{HOMO} + E_{LUMO}) η = - \frac{1}{2} (E_{HOMO} - E_{LUMO})$ $δ = \frac{1}{η} ω = \frac{χ^{2}}{2 η}$

As shown in Table 1, from the electronegativity (χ) and global softness (δ) values, the derivative 6b has minimum the Lewis’s acid character and the maximum charge transfer ability, respectively. Thus, the examined derivatives were sorted, according to softness, as 6b > 4b > 6a = 6c > 4a = 4c. Furthermore, the molecule dipole moment (μ) accounts the intermolecular interactions where the higher the dipole moment, the stronger the intermolecular interactions will be. The dipole moments of the studied compounds were ranged from 0.86 to 8.17 D for compound 6b and 4c, respectively.

3.2.2

3.2.2 Atomic Mulliken’s charges and Fukui’s indices

The Mulliken’s atomic charges shade light on the charge transfer and electronegativity properties of the molecule (Bhagyasree, Varghese et al. 2013). In all compounds, the pyrazolopyrimidine nitrogen atom N⁴_(Pp) has positive charge, 0.077–0.084. In contrast, the N¹_(Pp) and N⁸_(Pp) have positive and negative charges, 0.045–0.098 and −0.031 - −0.082, respectively, in 4a-c derivatives, while they possessed the opposite charges in 6a-c derivatives. Noteworthy, the pyrazolopyrimidine carbon atom C⁷_(Pp) in the cyano derivatives, 4a-c, have negative charge, −0.332 - −0.361, while in carboxamide derivatives, 6a-c, it is positively charged, 0.161–0,266. Thus, this behavior may be attributed to the electron withdrawing effect of the cyano group in comparison to the carboxamide group. In contrary, the nitrogen atoms of the cyano and amine substituents in addition to the oxygen atoms of the carboxamide groups were negatively charged whereas the nitrogen atoms of both phenylamine and carboxamide in all compounds were acquired positive charge (Table 2).

Table 2 The atomic Mulliken’s charges of investigated compounds.

Atom	4a	4b	4c	6a	6b	6c
N¹_(Pp)	0.082	0.098	0.045	−0.020	−0.019	−0.029
C²_(Pp)	−0.538	−0.544	−0.515	−0.544	−0.535	−0.522
C³_(Pp)	0.124	0.068	0.112	0.166	0.086	0.133
C^3a_(Pp)	−0.484	−0.444	−0.498	−0.581	−0.539	−0.599
N⁴_(Pp)	0.079	0.077	0.079	0.080	0.083	0.084
C⁵_(Pp)	−0.111	−0.111	−0.114	−0.099	−0.069	−0.102
C⁶_(Pp)	0.701	0.719	0.724	0.626	0.714	0.620
C⁷_(Pp)	−0.344	−0.332	−0.361	0.254	0.161	0.266
N⁸_(Pp)	−0.062	−0.082	−0.031	0.015	0.019	0.022
NH_(Pham)	0.138	0.145	0.139	0.124	0.136	0.129
C¹_(Pham)	0.038	0.017	0.059	0.130	0.078	0.158
C⁴_(Pham)	−0.256	−0.255	−0.264	−0.263	−0.488	−0.272
CO_(carb)	−0.360	−0.326	−0.385	−0.417	−0.345	−0.403
OC_(carb)	−0.203	−0.203	−0.199	−0.207	−0.208	−0.205
NH_(carb)	0.143	0.133	0.119	0.128	0.127	0.115
C¹_(Phcarb)	−0.336	−0.538	−0.589	−0.267	−0.487	−0.545
C⁴_(Phcarb)	0.673	−0.737	0.413	0.685	−0.718	0.406
NH_2(Pp)	−0.297	−0.297	−0.294	−0.426	−0.426	−0.425
CN_(Pp)	−0.895	−0.915	−0.907
NC_(Pp)	−0.163	−0.162	−0.160
CO_(carb1)				−0.435	−0.492	−0.450
OC_(carb1)				−0.316	−0.318	−0.315
NH_2(carb1)				−0.382	−0.387	−0.386
Me_(Phcarb)	−0.702	−0.527		−0.690
OMe_(Phcarb)		−0.030			−0.032
Cl_(Phcarb)			0.536			0.533

Moreover, the Fukui’s indices ( $f_{k}^{+}$ ), as a measure of the atomic reactivity toward nucleophilic attack (El Adnani, Mcharfi et al. 2013, Mi, Xiao et al. 2015, Olasunkanmi, Obot et al. 2016, Messali, Larouj et al. 2018 ) (Table S4), revealed similar patterns, e.g., the derivatives 4a-c showed that cyano nitrogen, NC_(Pp), followed by the pyrazolopyrimidine carbon at positions 5 and 3a, C⁵_(Pp) and C^3a_(Pp), while in 6a-c, the carbon of the fused ring at position 5, C⁵_(Pp), occupied the top site followed by oxygen and carbon of the carboxamide group, OC_(carb1) and CO_(carb1), respectively (Table 3). Alternatively, the electrophilic attack Fukui’s indices ( $f_{k}^{-}$ ) presented close patterns for the highly susceptible atoms, e.g., in derivatives 4a and 6a, the carbon 4 and nitrogen atoms of the phenylamine group, C⁴_(ph) and NH_(Pham), were occupied the second and third positions after cyano nitrogen atom, NC_(Pp), in derivative 4a while both were on the top in in derivative 6a, respectively. Whereas, the methoxy derivatives 4b and 6b showed that the methoxy oxygen, OMe_(ph), occupied the first place while the C¹_(ph) and C⁴_(ph) atoms became the second and third susceptible sites, respectively. Lastly, the indices of radical attack ( $f_{k}^{0}$ ) offered varied patterns, where in 4a-b, the cyano nitrogen, NC_(Pp), was on the top followed by the carbon 5 of the pyrazolopyrimidine, C⁵_(Pp), whereas, it occupied the first place in 6a-b before the carboxamide oxygen, OC_(carb1) (Table 3).(See Table 4).

Table 3 Selected top four atomic Fukui’s indices, the local relative electrophilicity and nucleophilicity descriptors data of the investigated compounds.

4a		4b		4c		6a		6b		6c
atom	$f_{k}^{-}$	atom	$f_{k}^{-}$	atom	$f_{k}^{-}$	atom	$f_{k}^{-}$	atom	$f_{k}^{-}$	atom	$f_{k}^{-}$
NC_(Pp)	0.058	OMe_(Phcarb)	0.064	C^l_(Phcarb)	0.077	C⁴_(Pham)	0.058	OMe_(Phcarb)	0.057	C^l_(Phcarb)	0.069
C⁴_(Pham)	0.055	C¹_(Phcarb)	0.050	C⁴_(Pham)	0.058	NH_(Pham)	0.056	C⁴_(Phcarb)	0.046	C⁴_(Pham)	0.061
NH_(Pham)	0.051	C⁴_(Phcarb)	0.050	NC_(Pp)	0.058	C³_(Pp)	0.037	C¹_(Phcarb)	0.042	NH_(Pham)	0.059
C⁴_(Phcarb)	0.040	NC_(Pp)	0.046	NH_(Pham)	0.054	C⁴_(Phcarb)	0.036	C³_(Phcarb)	0.040	C³_(Pp)	0.038
C³_(Pp)	0.033	C³_(Pham)	0.045	C³_(Pp)	0.035	C²_(Pham)	0.033	C²_(Phcarb)	0.037	C²_(Pham)	0.035
atom	$f_{k}^{+}$	atom	$f_{k}^{+}$	atom	$f_{k}^{+}$	atom	$f_{k}^{+}$	atom	$f_{k}^{+}$	atom	$f_{k}^{+}$
NC_(Pp)	0.110	NC_(Pp)	0.112	NC_(Pp)	0.106	C⁵_(Pp)	0.085	C⁵_(Pp)	0.085	C⁵_(Pp)	0.084
C⁵_(Pp)	0.098	C⁵_(Pp)	0.099	C⁵_(Pp)	0.097	OC_(carb1)	0.075	OC_(carb1)	0.076	OC_(carb1)	0.071
C^3a_(Pp)	0.051	C^3a_(Pp)	0.052	C^3a_(Pp)	0.050	CO_(carb1)	0.059	CO_(carb1)	0.060	CO_(carb1)	0.055
CN_(Pp)	0.045	CN_(Pp)	0.047	OC_(carb)	0.046	C^3a_(Pp)	0.046	C^3a_(Pp)	0.046	C^3a_(Pp)	0.046
N⁴(_Pp)	0.044	N⁴(_Pp)	0.045	Cl_(Phcarb)	0.045	N⁴(_Pp)	0.043	N⁴(_Pp)	0.045	Cl_(Phcarb)	0.043
atom	$f_{k}^{0}$	atom	$f_{k}^{0}$	atom	$f_{k}^{0}$	atom	$f_{k}^{0}$	atom	$f_{k}^{0}$	atom	$f_{k}^{0}$
NC_(Pp)	0.084	NC_(Pp)	0.079	NC_(Pp)	0.082	C⁵_(Pp)	0.054	C⁵_(Pp)	0.052	Cl_(Phcarb)	0.056
C⁵_(Pp)	0.061	C⁵_(Pp)	0.059	Cl_(Phcarb)	0.061	OC_(carb1)	0.051	OC_(carb1)	0.049	C⁵_(Pp)	0.054
C⁴_(Pham)	0.039	OMe_(Phcarb)	0.040	C⁵_(Pp)	0.060	C⁴_(Pham)	0.041	CO_(carb1)	0.035	OC_(carb1)	0.049
OC_(carb)	0.038	OC_(carb)	0.039	C⁴_(Pham)	0.041	C³_(Pp)	0.037	OC_(carb)	0.035	C⁴_(Pham)	0.042
C³_(Pp)	0.037	C⁴_(Phcarb)	0.034	OC_(carb)	0.038	CO_(carb1)	0.035	OMe_(Phcarb)	0.035	C³_(Pp)	0.037
atom	S⁺/S^-	atom	S⁺/S^-	atom	S⁺/S^-	atom	S⁺/S^-	atom	S⁺/S^-	atom	S⁺/S^-
C⁵_(Pp)	4.08	C⁵_(Pp)	5.50	CO_(carb)	4.57	CO_(carb1)	4.92	CO_(carb1)	6.00	CO_(carb1)	4.58
C^3a_(Pp)	3.64	C^3a_(Pp)	4.73	C⁵_(Pp)	4.04	C⁵_(Pp)	3.70	C⁵_(Pp)	4.72	CO_(carb)	4.14
CO_(carb)	3.38	C²_(Pp)	4.40	C^3a_(Pp)	3.57	CO_(carb)	3.57	C^3a_(Pp)	3.83	C⁵_(Pp)	3.65
CN_(Pp)	3.00	CN_(Pp)	4.27	CN_(Pp)	2.80	C^3a_(Pp)	3.29	C²_(Pp)	3.67	C^3a_(Pp)	3.29
N¹_(Pp)	2.50	N⁴_(Pp)	4.09	N¹_(Pp)	2.38	OC_(carb1)	2.78	N⁴_(Pp)	3.46	OC_(carb1)	2.73
atom	S⁺/S^-	atom	S⁺/S^-	atom	S⁺/S^-	atom	S⁺/S^-	atom	S⁺/S^-	atom	S⁺/S^-
C¹_(Phcarb)	9.50	C¹_(Phcarb)	12.50	C¹_(Phcarb)	15.00	C²_(Pham)	11.00	C¹_(Phcarb)	10.50	C²_(Pham)	11.67
C²_(Pham)	7.75	C²_(Phcarb)	5.13	C²_(Pham)	8.25	C¹_(Pham)	7.67	C²_(Pham)	6.67	C¹_(Phcarb)	11.00
C¹_(Pham)	7.00	OMe_(Phcarb)	4.27	C¹_(Pham)	5.75	C¹_(Phcarb)	7.00	C²_(Phcarb)	4.63	C¹_(Pham)	8.33
NH_(Pham)	4.25	C²_(Pham)	4.25	NH_(Pham)	4.91	NH_(Pham)	5.09	OMe_(Phcarb)	4.07	NH_(Pham)	5.90
C²_(Phcarb)	3.00	C⁶_(Phcarb)	3.89	C⁶_(Pham)	3.20	C⁶_(Pham)	3.30	C⁶_(Phcarb)	3.88	C⁶_(Pham)	3.50

Table 4 Cytotoxic activity of synthesized and designed compounds against human tumor cell lines.

Compound	Cytotoxicity IC₅₀ (μM)
Compound	Hep-2	HepG2	PC3	MCF-7	WI38
4a	23.12 ± 0.45	25.61 ± 0.21	34.44 ± 0.53	29.42 ± 0.23	26.15 ± 0.02
4b	19.82 ± 0.17	28.65 ± 0.43	37.64 ± 0.16	25.68 ± 0.16	34.64 ± 0.39
4c	16.78 ± 0.28	19.62 ± 0.13	29.36 ± 0.29	22.36 ± 0.23	35.18 ± 0.26
6a	12.76 ± 0.16	31.41 ± 0.05	26.19 ± 0.34	19.84 ± 0.49	32.47 ± 0.31
6b	10.71 ± 0.39	29.73 ± 0.66	24.90 ± 0.64	16.28 ± 0.11	37.26 ± 0.43
6c	8.85 ± 0.24	26.81 ± 0.26	26.37 ± 0.29	10.80 ± 0.36	42.64 ± 0.53
5-Fu	7.19 ± 0.47	5.33 ± 0.36	8.54 ± 0.23	10.19 ± 0.42	84.93 ± 0.28

5-Fluorouracil (5-Fu) is the known drug for antitumor assessments.

As the Fukui’s indices may be inaccurately predicted the active site for nucleophilic and electrophilic attack, the relative electrophilicity and nucleophilicity descriptors, $s_{k}^{-} / s_{k}^{+}$ and $s_{k}^{+} / s_{k}^{-}$ , respectively, were calculated (Roy, de Proft et al. 1998, Roy, Krishnamurti et al. 1998, Roy, Pal et al. 1999 ), where $s_{k}^{+} = f_{k}^{+} \times δ$ , $s_{k}^{-} = f_{k}^{-} \times δ$ and δ is global softness (Table S4). The relative nucleophilicity descriptors data, $s_{k}^{+} / s_{k}^{-}$ , presented resemble patterns, i.e., the 4a-c derivatives exhibited the C⁵_(Pp) at the top position except 4c in which the CO_(carb) was the first. While, the 6a-c derivatives displayed the CO_(carb1) as the most active site followed by the C⁵_(Pp) (Table 3). As well, the relative electrophilicity descriptors, $s_{k}^{-} / s_{k}^{+}$ , displayed that the phenyl carboxamide carbon C¹_(Phcarb) atom was the most active site in all derivatives except 6a and 6c in which the phenylamine carbon C²_(Pham) was on the top. The second position was occupied by difference atoms, i.e., the phenylamine carbon C²_(Pham) presented in 4a, 4c and 6b, while the 4b, 6a and 6c have the C²_(Phcarb), C¹_(Pham) and C¹_(Phcarb), respectively (Table 3).

3.3

3.3 Biological evaluation

3.3.1

3.3.1 In vitro anticancer activity

In comparison to 5-fluorouracil (5-Fu), the cytotoxicity of six pyrazolo[1,5-a]pyrimidine compounds was examined across four cancer cell lines HepG2, Hep-2, PC3, MCF-7, and normal fibroblast cells (WI38) (Abd El-Meguid, Awad et al. 2019). Fig. 5 reflected the variations of cytotoxicity in expression of IC₅₀. Inspired the structural lineaments associated to anticancer activity are shown by the results of the investigated pyrazolopyrimidine compounds. The synthesized pyrazolo[1,5-a]pyrimidines revealed as a whole respectable cytotoxic effectiveness with distinct reactivity to hinder the growing of MCF-7 rather than Hep-2. Initially, 7-amino-N-aryl-6-cyano-2-(phenylamino)-pyrazolo[1,5-a]pyrimidine-3-carboxamide analogues 4a-c were exhibited sensible inhibition activities toward MCF-7 (IC₅₀ lies between 22.36 ± 0.23–29.42 ± 0.23 μM) than Hep-2 (IC₅₀ lies between 16.78 ± 0.28–23.12 ± 0.45 μM), HepG2 (IC₅₀ lies between 19.62 ± 0.13–25.61 ± 0.21 μM), and PC3 (IC₅₀ lies between 29.36 ± 0.29–34.44 ± 0.53 μM) cell lines. Meanwhile, 7-Amino-2-(phenylamino)-pyrazolo[1,5-a]pyrimidine-3,6-dicarboxamides 6a-c were displayed respectable cytotoxic effectiveness toward MCF-7 (IC₅₀ lies between 10.80 ± 0.36–19.84 ± 0.49 μM) than Hep-2 (IC₅₀ lies between 8.85 ± 0.24–12.76 ± 0.16 μM), HepG2 (IC₅₀ lies between 26.81 ± 0.26–31.41 ± 0.05 μM), and PC3 (IC₅₀ lies between 24.90 ± 0.29–26.90 ± 0.34 μM) cell lines.

The interaction between hybrid 4c with (PDB ID: 5IVE).

3.3.2

3.3.2 Structural activity relationship

Rendering to cytotoxic outcomes of the prepared 7-amino-N-aryl-6-cyano-2-(phenylamino)-pyrazolo[1,5-a]pyrimidine-3-carboxamide analogues 4a-c, it was perceived that 2-(phenylamino)-pyrazolo[1,5-a]pyrimidine-6-carbonitrile hybrids presented high grade of reactivity towards adenocarcinoma cell line (MCF-7) (Rahmouni, Souiei et al. 2016). However, hybrid 4c (Ar = 4-ClC₆H₄) showed reasonable cytotoxic effectiveness with IC₅₀ = 22.36 ± 0.23 μM toward against MCF-7 cell line, more than compound 4b (Ar = 4-MeOC₆H₄) which demonstrated workable inhibition with IC₅₀ = 25.68 ± 0.16 μM. However, the hybrid 4a (Ar = 4-MeC₆H₄) revealed a little inhibition across MCF-7 with IC₅₀ = 29.42 ± 0.23 μM. Meanwhile, the previous survey offered that 2-(phenylamino)-pyrazolo[1,5-a]pyrimidine-3-carboxamides displayed substantial effectiveness over (MCF-7) (Arias-Gómez, Godoy et al. 2021). Moreover, 7-Amino-2-(phenylamino)-pyrazolo[1,5-a]pyrimidine-3,6-dicarboxamide hybrid 6c (Ar = 4-ClC₆H₄) demonstrated eminent effectiveness across MCF-7 with IC₅₀ = 10.80 ± 0.36 μM. Furthermore, hybrid 6b (Ar = 4-MeOC₆H₄) released pretty effectiveness with IC₅₀ = 16.28 ± 0.11 μM, then hybrid 6a (Ar = 4-MeC₆H₄) showed acceptable effectiveness across MCF-7 with IC₅₀ = 19.84 ± 0.49 μM. In accordance to the literature survey, the order of anti-tumor effectiveness follows the arranging of hybrids to these findings: 4-OCH₃ > 4-Cl > 4-CH₃ (Abbas and Abd El-Karim 2019, Al‐Anazi, Mahmoud et al. 2019). Additional examination of the prepared pyrazolo[1,5-a]pyrimidine hybrids was accomplished to realize their cytotoxic effectiveness on the normal cell fibroblast cells (WI38).

3.3.3

3.3.3 Molecular docking study

According to the scientific literature survey, new pyrazolo[1,5-a]pyrimidines have shown promise as KDM5A “histone lysine demethylase” inhibitors applied to treat breast cancer cells (Gehling, Bellon et al. 2016). These pyrazolo[1,5-a]pyrimidines possess structure like to ligand active site 5IVE. The finest structure of little tested compounds and the conformation of the KDM5A protein, that was recognized from (PDB- ID: 5IVE), were docked together (Metwally, Mohamed et al. 2019). The technique of docking progression was passed out to normalize the compound's preferred style of interrelating with the enzyme's active site. Meanwhile, the binding score (S, Kcal/mol) was utilized to compute the binding similarity of ligand over the enzyme active site; a low score of energy shows a good affinity. Through several prepared derivatives as a ligand and proteins as a target in a molecular docking simulation, the target-ligand interaction was located. The molecular docking study that was presented in Table 5, 7-amino-N-aryl-6-cyano-2-(phenylamino)-pyrazolo[1,5-a]pyrimidine-3-carboxamide analogues 4a-c displayed adequate binding score S = -7.4318, −7.5101, and −7.9031 Kcal/mol, respectively (Table 5). Hybrids 4a and 4b exhibited the same H- acceptor bonds between Asn 493 with N-atom of nitrile moiety over intermolecular distances (3.63 and 3.39 Å) (Figures S1 and S2). Meanwhile, hybrid 4c represented two H-bonds (H-donor and H-acceptor), Cl- atom with Leu 532 through (3.41 Å), N- atom of nitrile moiety with Asn 493 through (3.62 Å) (Fig. 5).

Table 5 Molecular docking data for the pyrazolo[1,5-a]pyrimidines analogues.

Code	S (Kcal/mol)	RMSD	Interaction with ligand	Types of Interactions	Distance (Å)
4a	−7.4318	1.0057	N- atom of nitrile moiety with Asn 493	H-acceptor	3.63
4b	−7.5101	0.9794	N- atom of nitrile moiety with Asn 493	H-acceptor	3.39
4c	−7.9031	0.9711	Cl- atom with Leu 532 N- atom of nitrile moiety with Asn 493	H-donor H- acceptor	3.41 3.62
6a	−7.0158	1.3333	O- atom of amide group with Asn 493	H-acceptor	2.99
6b	−7.6607	1.2059	N- atom of amide moiety with Ser 491 O- atom of amide moiety with Asn 493	H-donor H-acceptor	2.91 3.04
6c	−8.0107	1.0928	N- atom of amide moiety with Ser 491 O- atom of amide moiety with Asn 493 Cl- atom with Leu 532	H-donor H-donor H-acceptor	2.89 3.30 3.00
5-Fu	−4.2410	0.9567	O-atom of carbonyl moiety with Lys 501 Pyrimidine ring with Phe 480	H-acceptor π-π	3.07 3.70

Meantime, 7-Amino-2-(phenylamino)-pyrazolo[1,5-a]pyrimidine-3,6-dicarboxamide derivatives 6a- 6c revealed decent binding score S = -7.0158, −7.6607, and −8.0107 Kcal/mol, respectively. Hybrid 6a displayed H- acceptor amongst O- atom of amide group with Asn 493 through (2.99 Å) (Figure S3). Meanwhile, compound 6b displayed two H-bonds, H-donor among N-atom of amino moiety with Ser 491 through (2.91 Å), and H-acceptor between O-atom of amide substituent with Asn 493 through (3.04 Å) (Figure S4). Moreover, compound 6c revealed two H-donors among O-atom of amide substituent with Asn 493 through (3.30 Å), N- atom of amide moiety with Ser 491through (2.89 Å), and one H-acceptor between Cl- atom with Leu 532 through (3.00 Å) (Fig. 6).

The interaction between hybrid 6c and (PDB ID: 5IVE).

Furthermore, 5-Fu presented H-acceptor among O-atom of carbonyl moiety with Lys 501 through (3.07 Å), and π-π attraction between pyrimidine ring with Phe 480 through (3.70 Å) through RMSD = 0.9567 with low score S = -4.2410 Kcal/mol (Figure S5).

Finally, the outcomes of molecular docking were presented the subsequent summaries: 1) the docking scores of the synthesized hybrids gave acceptable values ranged between −7.0158 to −8.0107 Kcal/mol. 2) 7-Amino-2-(phenylamino)- pyrazolo[1,5-a]pyrimidine-3,6-dicarboxamide hybrids 6a-c were unveiled the furthermost many types of bindings like H-donor, H-acceptor, and π-π that offered superior confirmation for the best binding with the diverse amino-acids of 5IVE. 3) The synthesized hybrids were attached with the same amino-acids of protein over dissimilar polar and non-polar amino-acids like “Asn 493, and Leu 532” which presented good evidence for binding processes. 4) The resulted pictures of 2D, 3D, and surface map were reflected clear images of the close relationship between the synthesized ligands and the different types of 5IVE amino-acids.

4 Conclusion

A series of 7-amino-N-aryl-2-(phenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide analogues 4 and 6 were obtained through cyclocondensation of 3-aminopyrzoles 2a-c with 2-((dimethylamino)methylene)-malononitrile and/or 2-cyano-3-(dimethylamino)acrylamide. The DFT optimized structure of the investigated compounds indicated that all have almost planar configuration. All derivatives have similar HOMO and LUMO shapes and thus close ΔE_H-L gap was displayed, 2.59–2.81 eV, obeying the order 4a = 4c > 6c ≈ 6a > 4b > 6b. As well, the prepared 2-(phenylamino)-pyrazolo[1,5-a]pyrimidine derivatives demonstrated appropriate cytotoxic effectiveness with dissimilar activities to inhibit the growth of MCF-7 and Hep-2 cancer cell lines. Mainly, 7-amino-N-aryl-6-cyano-2-(phenylamino)-pyrazolo[1,5-a]pyrimidine-3-carboxamide analogues 4a-c were revealed respectable cytotoxic effectiveness toward MCF-7 rather than the rest of cell lines through IC₅₀ values (22.36 ± 0.23–29.42 ± 0.23 μM). Meanwhile, 7-Amino-2-(phenylamino)-pyrazolo[1,5-a]pyrimidine-3,6-dicarboxamide hybrids 6a-c were revealed attentive inhibition over IC₅₀ values against MCF-7 (10.80 ± 0.36–19.84 ± 0.49 μM) and Hep-2 (24.90 ± 0.29–26.90 ± 0.34 μM). All of the synthesized 2-(phenylamino)-pyrazolo[1,5-a]pyrimidine analogues were examined toward standard drug (5-Fu) with (IC₅₀ = 7.19 ± 0.47 μM) for Hep-2 and (IC₅₀ = 10.19 ± 0.42 μM) for MCF-7 in diminish to (WI38) normal cell. Moreover, the hypothetical outcomes of the molecular docking delivered acceptable approve over the findings of cytotoxic activity through the nominated (PDB Code-5IVE).

Acknowledgement

The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through Small Groups. Projects under grant number (RGP.1/102/43).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Appendix A

Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2022.104437.

Appendix A

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[1] Abbas H.A.S., Abd El-Karim S.S., . Design, synthesis and anticervical cancer activity of new benzofuran–pyrazol-hydrazono-thiazolidin-4-one hybrids as potential EGFR inhibitors and apoptosis inducing agents. Bioorg. Chem.. 2019;89:103035
[Google Scholar]

[2] Abd El-Meguid E., Awad H., Anwar M., . Synthesis of new 1,3,4-oxadiazole-benzimidazole derivatives as potential antioxidants and breast cancer inhibitors with apoptosis inducing activity. Russ. J. Gen. Chem.. 2019;89(348):356.
[Google Scholar]

[3] Al-Anazi K.M., Mahmoud A.H., AbulFarah M.A.A., . 2-Amino-5-arylazothiazole-based derivatives. in vitro cytotoxicity, antioxidant properties, and bleomycin-dependent DNA damage. ChemistrySelect. 2019;4:5570-5576.
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[4] Al-Azmi A., . Pyrazolo[1,5-a]pyrimidines: a close look into their synthesis and applications. Curr Org. Chem.. 2019;23(721):743.
[Google Scholar]

[5] Alcolea Palafox M., . Anticancer drug IUdR and other 5-halogen derivatives of 2′-deoxyuridine: conformers, hydrates, and structure–activity relationships. Struct. Chem.. 2014;25(53):69.
[Google Scholar]

[6] Ali G.M., Ibrahim D.A., Elmetwali A.M., . Design, synthesis and biological evaluation of certain CDK2 inhibitors based on pyrazole and pyrazolo[1,5-a]pyrimidine scaffold with apoptotic activity. Bioorg. Chem.. 2019;86(1):14.
[Google Scholar]

[7] Almehmadi S.J., Alsaedi A.M., Harras M.F., . Synthesis of a new series of pyrazolo[1,5-a]pyrimidines as CDK2 inhibitors and anti-leukemia. Bioorg. Chem.. 2021;117:105431
[Google Scholar]

[8] Arias-Gómez A., Godoy A., Portilla J., . Functional pyrazolo[1,5-a]pyrimidines: current approaches in synthetic transformations and uses as an antitumor scaffold. Molecules. 2021;26:2708.
[Google Scholar]

[9] Asano T., Yamazaki H., Kasahara C., . Identification, synthesis, and biological evaluation of 6-[(6R)-2-(4-Fluorophenyl)-6-(hydroxymethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-3-yl]-2-(2-methylphenyl) pyridazin-3(2 H)-one (AS1940477), a Potent p38 MAP kinase inhibitor. J. Med. Chem.. 2012;55(7772):7785.
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[20] El Adnani Z., Mcharfi M., Sfaira M., . DFT theoretical study of 7-R-3methylquinoxalin-2 (1H)-thiones (RH;CH3;Cl) as corrosion inhibitors in hydrochloric acid. Corros. Sci.. 2013;68(223):230.
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[Google Scholar]

[29] Jubeen F., Iqbal S.Z., Shafiq N., . Eco-friendly synthesis of pyrimidines and its derivatives: a review on broad spectrum bioactive moiety with huge therapeutic profile. Synth. Commun.. 2018;48(601):625.
[Google Scholar]

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[33] Lunagariya M.V., Thakor K.P., Waghela B.N., . Design, synthesis, pharmacological evaluation and DNA interaction studies of binuclear Pt (II) complexes with pyrazolo [1,5-a] pyrimidine scaffold. Appl. Organomet. Chem.. 2018;32:e4222.
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[Google Scholar]

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[Google Scholar]

[37] Matyugina E.S., Logashenko E.B., Zenkova M.A., . 5′-Norcarbocyclic analogues of furano [2,3-d] pyrimidine nucleosides. Heterocycl. Commun.. 2015;21(259):262.
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[39] Metwally N.H., Mohamed M.S., Ragb E.A., . Design, synthesis, anticancer evaluation, molecular docking and cell cycle analysis of 3-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidine derivatives as potent histone lysine demethylases (KDM) inhibitors and apoptosis inducers. Bioorg. Chem.. 2019;88:102929
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[40] Mi H., Xiao G., Chen X., . Theoretical evaluation of corrosion inhibition performance of three antipyrine compounds. Comput. Theor. Chem.. 2015;1072(7):14.
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Synthesis, anticancer evaluation, and molecular modeling study of new 2-(phenylamino)pyrazolo[1,5-a]pyrimidine analogues

Abstract

Keywords

3-aminopyrazoles

Pyrazolopyrimidines

Molecular modelling

MCF-7

Molecular docking

1 Introduction

2 Experimental

2.1 General remarks

2.2 Preparation of 3-amino-N-aryl-5-(phenylamino)-1H-pyrazole-4-carboxamide analogues 2a-c

2.3 Preparation of 7-amino-N-aryl-6-cyano-2-(phenylamino)-pyrazolo[1,5-a]pyrimidine-3-carboxamide analogues 4a-c

2.4 Synthesis of 7-amino-N3-aryl-2-(phenylamino)-pyrazolo[1,5-a]pyrimidine-3,6-dicarboxamide analogues 6a-c

2.5 Computational studies

2.6 MTT assay

2.7 Molecular docking study

3 Results and discussion

3.1 Preparation of 2-(phenylamino)pyrazolo[1,5-a]pyrimidine analogues 4 and 6

3.2 DFT molecular modeling study

3.2.1 Frontier molecular orbitals

3.2.2 Atomic Mulliken’s charges and Fukui’s indices

3.3 Biological evaluation

3.3.1 In vitro anticancer activity

3.3.2 Structural activity relationship

3.3.3 Molecular docking study

4 Conclusion

Acknowledgement

Declaration of Competing Interest

References

Appendix A

Supplementary material

Appendix A

Supplementary material

Supplementary data 1

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2.4 Synthesis of 7-amino-N₃-aryl-2-(phenylamino)-pyrazolo[1,5-a]pyrimidine-3,6-dicarboxamide analogues 6a-c