Synthesis of functionalized aminopyrazole and pyrazolopyrimidine derivatives: Molecular modeling and docking as anticancer agents

2.1 Chemistry

2.2 Computational studies

The obtained aminopyrazole and pyrazolopyrimidine derivatives were studied using the DFT/B3LYP/6–311⁺⁺G (Becke, 1993, Lee et al., 1988, Perdew and Wang, 1992) incorporated in Gaussian 09 W program (Frisch et al., 2009). The optimized geometries of all derivatives exhibited positive frequencies that cleared its stability. The Fukui indices were determined by Materials studio package DMol3 module (BIOVIA, 2017) utilizing the GGA and B3LYP functional with DNP (version 3.5) (Delley, 2006).

2.3 Cytotoxicity assay

Three individual cancer cell lines HepG2, MCF-7, and HCT-116, were examined toward in vitro cytotoxicity over the established MTT technique (Rahmouni et al., 2016) (as shown in the supplementary file).

2.4 Molecular docking (MD)

Using MOE, the most stable conformers of the synthetic analogues were constructed in 2D, 3D, and surface maps (software 2015.10). Lowest energy conformers of the synthetic analogues were docked into PDB: 5IVE. Protein's binding pocket from the Data Bank (Metwally et al., 2019). After the hydrogens were introduced, the enzyme structure was enhanced using a technique that gradually reduced and raised the restrictions on the enzyme until the RMSD gradient was lower than 1.5.

3.1 Chemistry

N-(4-Chlorophenyl)-2-oxo-2-(phenylamino)acetohydrazonoyl cyanide (3) has been prepared in the light of the reported literature by diazo-coupling of diazotized 4-chloroaniline with 2-cyanoacetanilide (Shawali and Abd El-Galil, 1971). Our synthetic strategy for producing many functionalized 4-aminopyrazole derivatives is based on the cyclization of N-(4-chlorophenyl)-2-oxo-2-(phenylamino)acetohydrazonoyl cyanide (3) with various alpha-halogenated reagents. Thus, heating in dioxane in the presence of triethylamine is required for the reaction of N-(4-chlorophenyl)-2-oxo-2-(phenylamino)-acetohydrazonoyl cyanide (3) with chloroacetone. The strong non-classical acidity of hydrazone-hydrogen (C⚌N-NH-) has been shown to be reactive in replacing chlorine from chloroacetone. The produced intermediate (A) undergoes intramolecular cyclization through nucleophilic addition of a methylene group on the nitrile function to produce the corresponding 5-acetyl-4-amino-pyrazole analogue 4 (Scheme 1). This analogue was utilized as a building block for the production of 7-methyl-pyrazolo[4,3-d]pyrimidine derivative 5 upon refluxing with phenyl isothiocyanate in pyridine. The compatible spectral and elemental analyses support the structures of the 4-aminopyrazole analogue 4 and the pyrazolo[4,3-d]pyrimidine derivative 5. The IR spectrum of 4-aminopyrazole 4 revealed the absorption frequencies of the –NH₂ and N—H functions at 3419, 3319, 3268 cm⁻¹. The absorption frequencies of the amidic carbonyl and the conjugated acetyl-carbonyl (intramolecular hydrogen bonded with the OH group) were observed at 1661 and 1597 cm⁻¹, respectively. The ¹H NMR signals of compound 4 are observed as a singlet at δ 2.10 ppm for the protons of methyl group (COCH₃), a singlet at δ 7.48 ppm for the protons of amino group (–NH₂), and a singlet at δ 9.78 ppm for the proton of the N—H group. The nine aromatic protons are observed as triplet, multiplet and doublet signals in the region δ 7.09–7.65 ppm.

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Scheme 1

Synthesis of 5-acetyl-4-aminopyrazole and pyrazolo[4,3-d]pyrimidine derivatives 4 and 5.

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In addition, treatment of N-(4-chlorophenyl)-2-oxo-2-(phenylamino)acetohydrazonoyl cyanide (3) with ethyl bromoacetate proceeded in refluxing dioxane and triethylamine to furnish the conforming 5-ethoxycarbonyl-4-amino-pyrazole analogue 6, which undergoes heating with phenyl isothiocyanate in pyridine to yield the corresponding 7-oxo-pyrazolo[4,3-d]pyrimidine derivative 7 (Scheme 2). The chemical structures of 4-aminopyrazole analogue 6 and its corresponding pyrazolo[4,3-d]pyrimidine derivative 7 were secured based on the compatible spectral and elemental analyses. The IR spectrum of pyrazolo[4,3-d]pyrimidine derivative 7 indicated the absorptions N—H groups at 3221 and 3183 cm⁻¹ in addition to a broad absorption for the carbonyl group at 1664 cm⁻¹. The ¹H NMR spectrum of compound 7 resonated as triplet, multiplet and doublet signals in the region δ 7.07–7.77 ppm for the aromatic protons and two singlet signals at δ 9.76 and 13.02 ppm for the protons of N—H groups. Furthermore, the reaction of N-(4-chlorophenyl)-2-oxo-2-(phenylamino)acetohydrazonoyl cyanide (3) with chloroacetonitrile in boiling dioxane and triethylamine afforded the targeting 4-amino-5-cyano-pyrazole analogue 8. The production of 7-imino-pyrazolo[4,3-d]pyrimidine derivative 9 has been achieved upon heating 4-amino-5-cyanopyrazole analogue 8 with phenyl isothiocyanate in dimethylformamide (DMF) at 85–90 °C for eight hours. The spectral and elemental analyses are used to elucidate the structure of aminopyrazole analogue 8 and its conforming pyrazolo[4,3-d]pyrimidine derivative 9.

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Scheme 2

Synthesis of 5-functionalized-4-aminopyrazoles 6 and 8, and their corresponding pyrazolo[4,3-d]pyrimidines 7 and 9.

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3.2 Molecular modeling features

The optimized structure of the 4-amino-1-(4-chlorophenyl)-N-phenyl-1H-pyrazole-3-carboxamide derivatives, 4 and 6, have a non-planar configuration in which the 4-chlorophenyl group was slanted on the pyrazolyl ring plane where the dihedral angle data disclosed that the C³_(Pyz)–N²_(Pyz)-N¹_(Pyz)-C¹_(PhPyz) and N²_(Pyz)-N¹_(Pyz)-C¹_(PhPyz)–C²_(PhPyz) were 173.0° and 45.2°, in the former, and −177.3° and 138.3°, in the latter, respectively. In contrary, the cyano analogue, derivative 8, has planar configuration, i.e., the corresponding angles were 180.0°. Furthermore, the phenyl carboxamide and amino groups in all derivatives was positioned in the pyrazole plane, where CO_(Carb)–C³_(Pyz)–N²_(Pyz)-N¹_(Pyz) = 179.5–180.0°, N²_(Pyz)–C³_(Pyz)–CO_(Carb)–NH_(Carb) = 0.0–1.1°, N²_(Pyz)–C³_(Pyz)–CO_(Carb)–OC_(Carb) = 178.8–180.0°, C³_(Pyz)–CO_(Carb)–NH_(Carb)-C¹_(PhCarb) = 179.8–180.0° and NH_2(Pyz)-C⁴_(Pyz)-C⁵_(Pyz)-N¹_(Pyz) = 178.7–180.0° (Fig. 2) (Table S1).

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Fig. 2

DFT Optimized structures of the 4, 6 and 8 derivatives.

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On the other hand, the 1-(4-chlorophenyl)-N,6-diphenyl-5-thioxo-5,6-dihydro-1H-pyrazolo[4,3-d]pyrimidine-3-carboxamide derivatives, 5, 7 and 8, have also a non-planar configuration where the former was more distorted than the others. The dihedral angle exhibited that each of pyrazolyl and pyrimidinyl rings, individually, were deviated from the planar structure, e.g., the C³_(Pyp)-C^3a_(Pyp)-C^7a_(Pyp)-N¹_(Pyp) and C^3a_(Pyp)-C^7a_(Pyp)-C⁷_(Pyp)-N⁶_(Pyp), in average, were 1.3° and 7.3°, while the joint between exhibited more distortion, e.g., the C7_(Pyp)-C^7a_(Pyp)-N¹_(Pyp)–N²_(Pyp) and C³_(Pyp)-C^3a_(Pyp)-C^7a_(Pyp)-C⁷_(Pyp), average, were 171.0° and 172.5°, respectively. The chlorophenyl moiety was differentially tilted on the pyrazolopyrimidine plane, e.g., in derivative 4, the C⁷_(Pyp)-C^7a_(Pyp)-N¹_(Pyp)-C¹_(PhPyp) and N²_(Pyp)-N¹_(Pyp)-C¹_(PhPyp)–C²_(PhPyp) were 22.7° and 37.0°, respectively, while the corresponding angles in derivative 7 were 0.6° and 174.1°, and in 9 were 13.5° and 137.6°, respectively. Similarly, the other phenyl ring, in position 6, was nearly vertical on the fused ring plane in all derivatives, i.e., C⁷_(Pyp)-N⁶_(Pyp)-C¹_(Ph)–C²_(Ph) has 92.2° average value. Moreover, both of the carbonyl carbon and phenyl ring of the phenyl carboxamide group were almost coplanar with the fused ring, e.g., CO_(Carb)–C³_(Pyp)–N²_(Pyp)-N¹_(Pyp) ≈ 178.8° and CO_(Carb)–NH_(Carb)-C¹_(PhCarb)–C²_(PhCarb) ≈ 179.4°, while both their amidic nitrogen and oxygen atoms were shifted out, e.g., N²_(Pyp)–C³_(Pyp)–CO_(Carb)–NH_(Carb) ≈ 157.5° and N²_(Pyp)–C³_(Pyp)–CO_(Carb)–OC_(Carb) ≈ 24.3°, respectively. Likewise, the thioxo sulfur atom were sited out the pyrazolopyrimidine plane by 5.0°, i.e., C⁷_(Pyp)-N⁶_(Pyp)-C⁵_(Pyp)-S_(Thio) ≈ 174.8°. Finally, the methyl, oxo and imino substituents in derivatives 5, 7 and 9, were roughly in plane with the fused ring, i.e., N¹_(Pyp)-C^7a_(Pyp)-C⁷_(Pyp)-Me_(Pyp), N¹_(Pyp)-C^7a_(Pyp)-C⁷_(Pyp)-O_(Oxo) and N¹_(Pyp)-C^7a_(Pyp)-C7_(Pyp)–NH_(Pyp) were 0.8°, −2.6° and 1.6°, respectively (Fig. 3) (Table S1).

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Fig. 3

DFT Optimized structures of the 5, 7 and 9 derivatives.

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Additionally, the DFT calculated bond lengths and angles were nearly coincided with those found in single crystal X-ray of similar compounds (Zhang et al., 2016, Şener et al., 2017, Wu et al., 2010) where lengths were longer by 0.11 Å maximum with 0.044–0.050 Å RMSD while the angles were deviated by 10.3° maximum with 5.34–6.91° RMSD, which could be attributed to the absence of intermolecular columbic interactions in the quantum chemical calculations as it carried out for isolated molecule in gaseous state, whilst the practical values were gotten for solid crystal lattice interacting molecules (Sajan et al., 2011) (Table S2-S3).

3.2.1

3.2.1 Frontier molecular orbitals

The molecule HOMO-LUMO construction and energetic values illuminate its electrons donation or receiving behavior (Bulat et al., 2004) as small HOMO-LUMO gap leads to ease intramolecular charge transfer (Xavier et al., 2015, Makhlouf et al., 2018) that may affect the molecule’s bioactivity (Bouchoucha et al., 2018). The 3D illustrations of the examined compounds FMO are presented in Figs. 3-4. The graph cleared that compound 4, 6 and 8 have HOMO spread over the whole molecule and consisted of the phenyl and pyrazole π-orbital with secondary participation of the heteroatom’s lone pairs. However, their LUMO was primarily built from the π*-orbitals of the whole molecule with very low contribution of the carboxamide phenyl ring. Thus, the HOMO-LUMO charge transfer may be mainly described as π → π* transition (Fig. 4).

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Fig. 4

The frontier molecular orbital of the compounds 4, 6 and 8.

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On contrary, the HOMO of 5, 7 and 9 derivatives were built mainly of the lone pair of electrons belong to the thioxo sulfur atom with minimum involvement of the π-orbital of the phenyl substituent on pyrazolopyrimidine moiety whereas their LUMO were constructed from the π*-orbital of the fused pyrazolopyrimidine ring. Therefore, the HOMO-LUMO charge transfer may be mainly described as n → π* transition (Fig. 5).

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Fig. 5

The frontier molecular orbital of the compounds 5, 7 and 9.

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The HOMO-LUMO energy, EH and EL, were influenced by the aforementioned details. Hence, the data revealed that the all derivatives have close values of the EH and EL, range −5.66 - −6.21 and −3.58 - −4.49 eV, respectively, where 5 and 6 exhibited lowest EH and EL, respectively. Additionally, the ΔE_H-L gap data indicated that 5 and both of 6 and 8 possessed the lowest and highest values, 1.16 and 2.35 eV, respectively. Accordingly, the studied derivatives could be ordered according to energy gap as 5 < 9 < 7 < 4 < 6 = 8 which clear that the pyrazolopyrimidine derivatives have lower energy gap than the corresponding pyrazoles (Table 1).

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

Compound	EH	EL	ΔEH-L	χ	η	δ	ω
4	−6.00	−3.76	2.25	4.88	1.12	0.89	10.60
5	−5.66	−4.49	1.16	5.08	0.58	1.72	22.13
6	−5.93	−3.58	2.35	4.75	1.18	0.85	9.60
7	−6.21	−4.07	2.13	5.14	1.07	0.94	12.39
8	−6.19	−3.84	2.35	5.01	1.17	0.85	10.71
9	−5.91	−3.80	2.10	4.85	1.05	0.95	11.22

Respectively, chemical reactivity descriptors, such as electronegativity (χ), global hardness (η), softness (δ) and electrophilicity (ω), were determined using the values of EH and EL by the following formulae (Xavier et al., 2015). $$\chi =-\frac{1}{2}\left(E_{\mathit{HOMO}}+E_{\mathit{LUMO}}\right)\eta =-\frac{1}{2}\left(E_{\mathit{HOMO}}-E_{\mathit{LUMO}}\right)$$ $$\delta =\frac{1}{\eta }\omega =\frac{{\chi }^2}{2\eta }$$

The data revealed that, according to electronegativity (χ), derivative 6 and 7 exhibited the lowest and highest Lewis’s acid character, 4.75 and 5.14 eV, respectively. Whereas, the global softness (δ) and hardness (η) values designated that compound 5 possessed the maximum electrons receiving and charge transfer ability, 1.72 and 0.58 eV, respectively. Hence, in consistent with hardness, the studied derivatives may be ordered as 5 < 9 < 7 < 4 < 8 > 6, in agreement with that of ΔE_H-L gap (Table 1).

3.3 Cytotoxicity assay

Six 4-aminopyrazole and pyrazolopyrimidine analogues were examined over MTT assay toward several cancer cell lines such as HCT-116 (colon carcinoma), HepG2 (liver carcinoma), MCF-7(breast carcinoma), and WI38 (normal cell) at the National Cancer Institute, (Egypt). Table 4 and Fig. 6 revealed the cytotoxic results in standings of IC₅₀ values and contrasted them to the extensively used anticancer reference (5-Fu). Some of the synthesized aminopyrazoles and pyrazolopyrimidines presented good efficiency to inhibit the growth of (MCF-7) and (HCT-116) rather than HepG2 (Fayed et al., 2019). Among the deliberate analogues, pyrazolopyrimidine analogues 5, 7, and 9 were presented extra cytotoxic activities especially against both of (MCF-7) and (HCT-116). The examined results of pyrazolopyrimidine analogues deliver understanding into the structural features related to anticancer activity. Initially, aminopyrazole analogue 4 have acetyl moiety in 5-position demonstrated reasonable activity in general with IC₅₀ = 23.42 ± 0.26, 27.25 ± 0.14, and 33.75 ± 0.42 μM against MCF-7, HCT-116, and HepG2, respectively. While, analogue 6 with ethyl ester group in 5-position offered weak activities with IC₅₀ = 34.51 ± 0.47, 39.76 ± 0.19, and 37.51 ± 0.32 μM against the same sequence of cell lines MCF-7, HCT-116, and HepG2, correspondingly. Further analogue 8 through nitrile group in 5-position presented enhancement of cytotoxic activity with IC₅₀ = 19.33 ± 0.18, 22.25 ± 0.53, and 18.42 ± 0.10 μM. However, pyrazolopyrimidine analogues such as analogue 5 with methyl group in 7-position displayed better antitumor activities linked to their substituents with IC₅₀ = 13.47 ± 0.25, 19.35 ± 0.27, and 14.82 ± 0.38 μM. Moreover, analogue 7 with a carbonyl moiety in 7-position exhibited eminent cytotoxic activity with IC₅₀ = 11.51 ± 0.35, 15.08 ± 0.27, and 21.25 ± 0.37 μM. Furthermore, analogue 9 with an imino moiety in 7-position revealed superior activities with IC₅₀ values 16.48 ± 0.36, 16.38 ± 0.27, and 17.39 ± 0.46 μM toward the same order of cell lines MCF-7, HCT-116, and HepG2, respectively. All of the synthesized analogues were studied in comparison to reference (5-Fu).

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Fig. 6

In vitro cytotoxicity of the prepared analogues.

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3.4 Structural activity relationship

Inspired the above results of the cytotoxic effectiveness, the six aminopyrazole and pyrazolopyrimidine analogues showed potent activities against (MCF-7, HCT-116) and less potent against HepG2 as it was reported before (Hassan et al., 2021). Among the amino-pyrazole analogues 4, 6, and 8 substituted with different acetyl, ester, and cyano moieties in 5-position, respectively, displayed various cytotoxic activities, analogue 4 have acetyl moiety demonstrated reasonable activity, analogue 6 with ethyl ester group offered weak activities, analogue 8 through nitrile group presented enhancement of cytotoxic activity. These results presented that, the cytotoxic activities follow the following order CN substituent > acetyl substituent > ester substituent which agrees with pervious literature (Farag et al., 2010). Meanwhile, pyrazolopyrimidine analogues were own broadly powerful than aminopyrazole as antitumor activity towards certain cancer cell lines (Hassan et al., 2020). Pyrazolopyrimidine analogues 5, 7, and 9 displayed significant anticancer activities toward MCF-7 (IC₅₀ = 13.47 ± 0.25, 11.51 ± 0.35, 16.48 ± 0.36), respectively, which is presented a potent activities than pervious Pyrazolopyrimidine derivatives that was recorded in the literature with (IC₅₀ = 13.91 ± 1.4 and 22.37 ± 1.8 μM/L) (Metwally et al., 2019). As analogue 7 have methyl group in 7-position showed high IC₅₀ value may attribute to the existence of hydrophobic group which increases the dual property lipophilic/hydrophilic part in the structure rather than both of 5 and 9 analogues with carbonyl and imino groups that possess a higher hydrophilic property (Wang et al., 2021).

Finally, the Multiple Linear Regression (MLR) (Worachartcheewan et al., 2011) incorporated in OriginPro software (OriginLab, 2018) was applied to correlate the anticancer activity (IC₅₀), dependent variable, with the descriptors of the quantum chemical calculation, E_H, E_L, ΔE_H-L, χ and η, independent variables, to find out the most effective descriptor on the anticancer activity (Table 5). The data indicated that all the cell lines, except WI38, exhibited close coefficients in which the most effective descriptor were the ΔE_H-L gap and hardness (η), both have positive values, and thus the increase in HOMO-LUMO gap and hardness will lead to increase in the activity. While the E_H, E_L, and χ displayed negative coefficients and consequently their increase will result in reduction of the anticancer activity. Moreover, these cell lines showed close intercept values, 263.96–284.46, and good regression coefficients, R² = 0.8593–0.9999 with standard deviation, SD = 0.14–6.75. On contrary, the normal cell, WI38, exhibited opposite behavior where the ΔE_H-L gap and hardness (η) were negative whereas the others have positive coefficients with regression coefficient, R² = 0.9854.

3.5 Molecular docking

A molecular docking simulation via utilizing MOE v10.2015.10 program was carried out to determine the compound's preferred manner of interacting with the enzyme's active site. Energy score (S, kcal/mol) was used to calculate the binding affinity of the synthesized derivatives with the enzyme active site; a low energy score implies a good affinity. The interaction types that are presented in molecular docking are hydrogen bonds, π-H, H-π, and π- π interactions, and arene cation interactions. By using several tested ligand and proteins as a target in a molecular docking simulation, the target-ligand interaction was determined. According to literature survey, it was noticed that PPs recorded a potent activity toward histone lysine demethylases (KDM) inhibitors (Metwally et al., 2019). The crystal structure of the KDM5A protein, which was determined from the protein data bank (PDB- ID: 5IVE), and the most stable conformation of certain investigated drugs were used to perform molecular docking to introduce a theoretical prediction for the reactivity of the synthesized derivatives towards (KDM) to help the biological scientists to investigate these test experimentally. The docking results (Table 6) displayed binding energy score (S), route main square (RMSD), types and site of interactions between ligands and 5IVE amino-acids, and the intermolecular distances (Å) for the synthesized analogues. 5-Acetyl-4-amino-1-(4-chlorophenyl)-N-phenyl-1H-pyrazole-3-carboxamide (4) displayed binding energy value S = −7.1126 kcal/mol, standing on RMSD = 1.4975 through two π-H interaction bindings, phenyl ring with both of His 483 and His 571 through 4.47 Å, 4.28 Å, respectively (Figure S1). Whereas, 1-(4-Chlorophenyl)-7-methyl-pyrazolo[4,3-d]pyrimidine-3-carboxamide analogue 5 revealed best RMSD = 0.8213 from one π- π interaction phenyl ring with His 483 through 3.91, with binding energy value S = -7.5571 kcal/mol (Fig. 7).

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Fig. 7

Docking interactions over pyrazolopyrimidine compound 5 with 5IVE.

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While, analogue 6 with amino and ester substituents on the pyrazole moiety showed RMSD = 1.4039 with binding energy value S = -7.5158 kcal/mol through H-acceptor between O24 of carbonyl ester with His 483 which form another π- π interaction between the phenyl-pyrazole ring through distances 2.95 and 3.77 Å (Figure S2). Likewise, analogue 7 revealed better binding score, S = -8.3527 kcal/mol with RMSD value = 1.0311 over H-π interaction between N 16 of amide linkage with His 483 through 3.65 Å (Fig. 8).

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Fig. 8

Docking interactions over pyrazolopyrimidine compound 7 with 5IVE.

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Also, analogue 8 showed lower value, S = -7.0005 kcal/mol with RMSD value = 1.0765 from H-acceptor raised between O9 of amide linkage with Lys 501, N 24 of nitrile moiety with His 571, and π- π interaction between the phenyl-pyrazole ring with His 483 through 3.46, 3.18, and 3.79 Å (Figure S3). Meanwhile, analogue 9 disclosed one H-acceptor between S7 of thio-emino moiety with Leu 536 through 3.25 Å, π-H interaction between the phenyl-pyrazole ring with His 571 through 4.88 Å, binding energy value S = -7.2823 kcal/mol with RMSD = 0.8924 (Figure S4). Similarly, 5-fluorouracil one H-acceptor between O3 of one of carbonyl group in the pyrimidinone ring with LYS 501 through 3.07 Å, π- π interaction between the pyrimidinone ring with Phe 480 through 3.70 Å, binding energy value S = -4.3533 kcal/mol with RMSD = 0.9599 (Figure S5).

Finally, the stimulating reasonable docking experiment is used to confirm how much the synthesized analogues interactions change as they interact with many analogues. A network of hydrogen bonding with the chemically aromatic moiety that interacts differently with the amino acids of 5IVE supports each of the six synthesized analogues that were created. The 5IVE pocket, which can be visualized as a fork enclosed by polar and nonpolar residues of amino-acids (His 483, His 571, Lys 501, Leu 536, Gly287) with the majority of the analogues that were prepared. Moreover, most of different derivatives are related to the same amino acid (s is considered as strong evidence for success docking procedure.