Synthesis of New Pyrazolo[3,4-d]pyrimidine Derivatives: NMR Spectroscopic Characterization, X-Ray, Hirshfeld Surface Analysis, DFT, Molecular Docking, and Antiproliferative Activity Investigations

Abstract

Four new pyrazolo[3,4-d]pyrimidines (P1–P4) were successfully synthesized in good relative yields by reacting 3-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-ol with various alkylating agents (methyl iodide, propargyl bromide, and phenacyl bromide) at room temperature in DMF solvent, employing liquid–solid phase transfer catalysis. The P1–P4 structures were elucidated using NMR spectroscopy and X-ray diffraction. Intermolecular interactions in P1–P4 were analyzed via Hirshfeld surface analysis and 2D fingerprint plots. Geometrical parameters were accurately modeled by DFT calculations using the B3LYP hybrid functional combined with a 6–311++G(d,p) basis set. The antiproliferative activity of P1–P4 towards colorectal carcinoma (HCT 116), human hepatocellular carcinoma (HepG2), and human breast cancer (MCF-7) cell lines, along with one normal cell line (WI38) was investigated using the MTT assay and sunitinib as a reference. Compounds P1 and P2 exhibited antiproliferative activities comparable to the reference drug towards all tested cells, with an IC₅₀ range of 22.7–40.75 µM. Both compounds also showed high selectivity indices and minimal cytotoxic effects on the normal cell line. Molecular docking revealed that the significant antiproliferative activity may attributed to the number and type of intermolecular hydrogen bonding established between pyrazolo[3,4-d]pyrimidines and DNA topoisomerase, a common target for various anticancer agents.

1. Introduction

Cancer is one of the foremost causes of the increase in the number of deaths and morbidity around the globe, mainly in the aging population, and appears to be more likely related to changes in unhealthy lifestyles. Various cancers are categorized according to the damaged tissue or organ. Certain cancers have well-defined mechanisms through which normal cells become cancerous. In the last decades of the 20th century, oncological studies reported that most cancerous cells exhibit a few acquired molecular, biochemical, and cellular characteristics that result from the alteration of key pathways [1]. Heterocycles are the main component of the widely available anti-cancer drugs; the FDA reported that two-thirds of anti-cancer agents contain heterocycle rings. In recent years, there has been increased interest in the chemistry of pyrazolo[3,4-d]pyrimidines owing to their wide-ranging biological activities, such as anticancer [2], antibacterial [3], antitubercular [4], and antitumor effects [5].

This nucleus constitutes a class of heterocyclic compounds of interest both chemically and pharmaceutically, as they were found to exhibit their antitumor activity by inhibiting different types of enzymes such as Bruton’s tyrosine kinase (BTK) [6], Src tyrosine kinase [7,8], Myt1 kinase [9], and TRAP1 kinase [10]. To the best of our knowledge, the first pyrazolo[3,4-d]pyrimidine kinase inhibitors identified were 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) (Figure 1), which were first discovered to act as kinase inhibitors of the SRC family of non-receptor tyrosine kinases in 1996 [11]. Indeed, pyrazolo[3,4-d]pyrimidines have several active sites, giving them a high reactivity, making these heterocycles excellent precursors in synthesizing new heterocyclic systems likely to present a broad spectrum of activity. For example, Allopurinol (Figure 1), first synthesized by Robins is used in human clinics as a drug in the treatment of gout, a condition characterized by increased levels of uric acid in the blood [12]. Accordingly, synthesizing new pyrazolo[3,4-d]pyrimidines has attracted more attention using various synthetic methods [13,14,15,16,17].

Given the importance that pyrazolo[3,4-d]pyrimidine derivatives brought to the biological and therapeutic fields and in continuation of our previous studies [18,19], we report the synthesis and structural elucidation of four new pyrazolo[3,4-d]pyrimidines (P1–P4) that have diverse groups at position 1 of the pyrazolopyrimidine core. Their structures were elucidated by X-ray crystallography and NMR spectroscopy. Additional studies of the new molecules included Hirshfeld surface analysis, DFT calculations, molecular docking, and in vitro antiproliferative investigations.

2. Results and Discussion

2.1. X-Ray Structure Descriptions

2.1.1. Crystal Structure of 3-methyl-1-phenyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (P1)

The pyrazolopyrimidine unit is slightly non-planar as indicated by the dihedral angle of 1.22(8)° between the mean planes of the constituent rings. The mean plane of the C6···C11 benzene ring is inclined to that of the pyrazole by a dihedral angle of 5.29(9)° so the whole molecular structure is nearly flat (Figure 2).

In the crystal, inversion dimers are formed by paired N1—H1···O1 hydrogen bonds (

Table 1. Hydrogen bond geometry (Å, °) for P1. Cg3 is the centroid of the C6···C11 benzene ring.

D—H···A	D—H	H···A	D···A	D—H···A
N1—H1···O1 i	0.917 (9)	1.877 (9)	2.7912 (13)	174.9 (14)
C2—H2···Cg3 ii	0.95	2.86	3.7328 (14)	154
C7—H7···N2	0.95	2.30	2.9746 (17)	127

Symmetry codes: (i) −x + 3, −y + 1, −z + 1; (ii) −x + 3/2, y + 1/2, −z + 1/2.

). The dimers are linked by C2—H2···Cg3 interactions (Table 1) to form corrugated layers of molecules parallel to ($10\overline{3}$) (Figure 3). These layers are connected along the a-axis direction through slipped π-stacking interactions between phenyl and pyrazole rings (centroid···centroid = 3.5730(8) Å, dihedral angle = 5.29(7)°) with the slippage alternating between 1.02 and 1.33 Å along the stack (Figure S1). Alternatively, the packing can be described as consisting of oblique stacks of molecules connected by the π-stacking interactions which are then joined by the C2—H2···Cg3 interactions. Crystal, data collection, and refinement details are presented in Table S1.

2.1.2. Crystal Structure of 3,5-dimethyl-1-phenyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (P2)

The pyrazolopyrimidine moiety is planar to within 0.0194(7) Å (rmsd = 0.0150 Å) with the plane of the C8···C13 ring inclined to it by 32.38(4)° (Figure 2). In the crystal, C5—H5···N2 hydrogen bonds (

Table 2. Hydrogen bond geometry (Å, °) for P2. Cg3 is the centroid of the C8···C13 benzene ring.

D—H···A	D—H	H···A	D···A	D—H···A
C5—H5···N2 i	0.95	2.57	3.4517 (17)	154
C6—H6A···Cg3 ii	0.98	2.92	3.5166 (19)	121
C9—H9···Cg3 iii	0.95	2.99	3.7890 (19)	142

Symmetry codes: (i) −x + 1, y + 1/2, −z + 3/2; (ii) −x + 1, −y + 1, −z + 2; (iii) x, −y − 1/2, z − 1/2.

) form chains of molecules extending along the b-axis direction and with the pyrazolopyrimidine moieties approximately parallel to ($10\overline{1}$). These chains stack along the normal to ($10\overline{1}$) with C6—H6A···Cg3 and C9—H9···Cg3 interactions (Table 2) holding them together to form layers (Figure 4). Crystal, data collection, and refinement details are presented in Table S2.

2.1.3. Crystal Structure of 3-methyl-1-phenyl-5-(prop-2-yn-1-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidinone (P3)

The dihydropyrazolopyrimidine moiety is not planar as there is a dihedral angle of 4.38(4)° between the mean planes of its constituent rings. The plane, defined by N2/C6/C7/C8, is inclined to the mean plane of the dihydropyrimidine ring by 84.66(8)° indicating the propynyl group is well out of the plane of the ring. The dihedral angle between the mean planes of the dihydropyrimidine and phenyl rings is only 4.38(5)° and this is likely due to the intramolecular C15—H15···N1 hydrogen bond (

Table 3. Hydrogen bonding geometry (Å, °) for P3.

D—H···A	D—H	H···A	D···A	D—H···A
C2—H2···O1 i	0.95	2.42	3.3115 (15)	156
C8—H8···N3 ii	0.95	2.48	3.3915 (18)	160
C15—H15···N1	0.95	2.28	2.9506 (17)	127

Symmetry codes: (i) x, −y + 1/2, z + 1/2; (ii) −x + 2, y − 1/2, −z + 1/2.

and Figure 2). In the crystal, zigzag chains of molecules extending along the c-axis direction are formed by C32—H2···O1 hydrogen bonds (Table 3) and are connected into corrugated layers two molecules thick by C8—H8···N3 hydrogen bonds (Table 3) and π-stacking interactions between phenyl and dihydropyrimidine rings in inversion-related molecules (centroid···centroid (at −x + 2, −y + 1, −z + 1) = 3.6770(8) Å, dihedral angle = 4.91(6)°, slippage = 1.19 Å) (Figure 5). The layers stack along the a-axis direction through π-stacking interactions between phenyl and dihydropyrimidine rings in inversion-related molecules (centroid···centroid (at −x + 1, −y + 1, −z + 1) = 3.7177(8) Å, dihedral angle = 4.91(6)°, slippage = 1.66 Å) (Figures S2 and S3). Data collection and refinement details are given in Table S3.

2.1.4. Crystal Structure of 3-methyl-5-(2-oxo-2-phenylethyl)-1-phenyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (P4)

The dihydropyrazolopyrimidine moiety is planar to within 0.0102(6) Å (r.m.s. deviation = 0.0087 Å). The mean planes of the C7···C12 and the C15···C20 benzene rings are inclined to the above plane by 29.25(3) and 85.79(3)°, respectively. The C1—N1—C13—C14 torsion angle of −78.99(9)° indicates that the C14=O2 carbonyl group is also nearly perpendicular to the mean plane of the dihydropyrazolopyrimidine moiety (Figure 2). In the crystal, C11—H11···O2 and C20—H20···O2 hydrogen bonds (

Table 4. Hydrogen bond geometry (Å, °) for P4. Cg4 is the centroid of the C15···C20 benzene ring.

D—H···A	D—H	H···A	D···A	D—H···A
C6—H6C···N2 i	0.96	2.56	3.5153 (12)	178
C9—H9···Cg4 i	0.93	2.68	3.5863 (12)	166
C11—H11···O2 ii	0.93	2.55	3.2895 (13)	137
C20—H20···O2 iii	0.93	2.55	3.2895 (13)	137

Symmetry codes: (i) −x + 1, y − 1/2, −z + 1/2; (ii) −x + 1, −y + 1, −z; (iii) −x, −y + 1, −z.

) form chains of molecules extending along the a-axis direction (Figure 6). The chains are linked by C6—H6C···N2 hydrogen bonds and C9—H9···Cg4 interactions (Table 4) forming layers approximately parallel to (011) (Figure S4). Crystal, data collection, and refinement details are presented in Table S4.

2.1.5. General Structural Comments

In all four structures, the sum of the angles about the nitrogen atom in the pyrazole ring bearing the phenyl substituent is 360° within experimental error, thereby implicating the involvement of the nitrogen lone pair in π bonding with neighboring atoms. As might be expected, this occurs within the five-membered ring. For example, in P3, The endocyclic N4—C1 and N4—N3 distances are, respectively, 1.3591(15) and 1.3805(14) Å, while the exocyclic N4—C10 distance is 1.4236(15) Å. Similar patterns are seen in the other structures. A second feature of note is the extent to which the plane of the pendant phenyl group is rotated from the plane of the five-membered ring to which it is attached. In the case of P3, it was noted that there is an intramolecular C—H···N hydrogen bond which likely constrains the phenyl group to approach co-planarity with the 5-membered ring but in the others, the dihedral angle is 5.29(9)° in P1, 32.38(4)° in P2, and 29.25(3)° in P4. A survey of 13 related molecules found corresponding dihedral angles ranging from 5.72(18)° in 5-(2-chloroethyl)-1-(4-chlorophenyl)-6-(2-chloropyridin-3-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one to 28.96(11)° in 5-(2-chloroethyl)-6-(3-fluorophenyl)-1-phenyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one [20], with the majority among the 13 structures surveyed being between 20 and 27°. Thus, it seems likely that packing considerations are predominant for determining this dihedral angle. If the strongest intermolecular interactions act to keep the dihydropyrazolopyrimidine cores well-separated, then the phenyl ring can rotate out of the plane of the core moiety to relieve intramolecular steric congestion. If not, π-stacking interactions can be influential which will necessitate the five- and six-membered rings being closer to co-planarity.

2.2. Optimized Geometries

Figure 7 shows the optimized geometries of P1–P4 along with their corresponding numbering and their superpositions upon the experimental structures. Selected experimental and calculated bond lengths, bond angles, and torsion angles for P1–P4 are summarized in

Table 5. Selected experimental and calculated geometrical parameters of P1–P4.

	P1			P2			P3			P4
	X-Ray	Cal	ΔX a	X-Ray	Cal	ΔX a	X-Ray	Cal	ΔX a	X-Ray	Cal	ΔX a
Bond length, B(Å)
C1-O1	1.239	1.224	0.01	1.220	1.225	0.00	1.225	1.223	0.00	1.220	1.225	0.00
C1-N3	1.392	1.418	0.03	1.407	1.427	0.02	1.417	1.431	0.01	1.424	1.430	0.01
N9-N10	1.387	1.376	0.01	1.378	1.377	0.00	1.381	1.376	0.00	1.385	1.376	0.01
N10-C12	1.425	1.425	0.00	1.415	1.424	0.01	1.424	1.424	0.00	1.420	1.424	0.00
N3-C18	-	-	-	1.465	1.468	0.00	1.479	1.477	0.00	1.458	1.457	0.00
C18-C19	-	-	-	-	-	-	1.466	1.462	0.00	1.528	1.538	0.01
C19-O20	-	-	-	-	-	-	-	-	-	1.215	1.216	0.00
Bond angles, A(°)
N3-C1-C7	111.90	110.46	1.45	111.81	111.65	0.16	111.25	111.24	0.01	111.23	111.43	0.20
N5-C6-N10	126.72	126.56	0.16	126.17	127.07	0.90	125.89	126.92	1.03	126.31	126.94	0.64
C1-N3-C18	-	-	-	117.27	117.01	0.26	117.96	117.75	0.21	117.10	117.52	0.42
N3-C18-C19	-	-	-	-	-	0.00	110.60	112.92	2.32	111.20	112.17	0.98
C18-C19-C20	-	-	-	-	-	0.00	176.61	179.33	2.72			0.00
C18-C19-O20	-	-	-	-	-	-	-	-	-	119.79	119.99	0.20
Torsion angles, D(°)
O2-C1-C7-C8	−2.35	1.19	3.54	2.63	1.26	1.37	−5.60	1.21	6.81	−0.20	−1.40	1.21
N5-C6-N10-C12	2.58	−0.12	2.71	0.09	−0.54	0.63	−0.15	−0.64	0.49	4.43	0.62	3.81
N9-N10-C12-C17	6.02	33.01	26.99	31.34	30.62	0.72	5.51	31.55	26.04	−27.06	−32.12	5.05
O2-C1-N3-C18	-	-	-	−2.24	0.02	2.26	3.72	0.96	2.75	−3.93	−2.66	1.27
C1-N3-C18-C19	-	-	-	-	-	0.00	82.62	91.88	9.26	−78.99	−90.41	11.41
N3-C18-C19-O20	-	-	-	-	-	-	-	-	-	5.07	−6.01	11.08
C18-C19-C21-C22	−2.35	1.19	3.54	2.63	1.26	1.37	−5.60	1.21	6.81	−0.20	−1.40	1.21

^a X = B, A or D, where B is for B (bond length), A for bond angle, and D for torsion angle.

. The experimental bond lengths are relatively well reproduced with variations less than 0.03 Å. Similarly, the bond angles are relatively well reproduced with variations of less than 3 degrees compared to the experimental ones. The agreement between calculated and experimental torsion angles is much poorer, reflecting the presence of packing constraints in the latter which are absent in the former. RMSD values calculated from the superimposed X-ray and optimized geometries of P1–P4 are 0.33, 0.087 Å, 0.43, and 0.63, respectively. Furthermore, the RMSD values were calculated for bond lengths, bond angles, and dihedral angles of P1–P4, and they are found in ranges of 0.08–0.12Å, 0.50–5.00 degrees, and 3.50–13.00 degrees, respectively. Figure S13 displays the linear regression curves between the experimental bond lengths, bond angles, and dihedral angles, and their corresponding predicted ones. The correlation between the experimental bond lengths, bond angles, and torsion angles with their corresponding calculated ones yield linear curves of correlation coefficients in the ranges of 92.00–99.50%, 91.50–99.95%, and 99.00–99.90%, respectively (Figure S13).

2.3. Hirshfeld Surface Analysis

The intermolecular bonding interactions in P1–P4 crystals were investigated by analyzing their corresponding Hirshfeld surfaces (HS). Descriptions and interpretations of the plots obtained from the HS analysis have been published [21]. The red spots on the Hirshfeld surface indicate contacts less than the sum of the van der Waals radii of the respective elements, while those longer than this sum are displayed in blue, and those on the order of this sum are shown in white (Figure 8, left column). The right column of Figure 8 shows each molecule with its HS surrounded by several “next neighbors” that are involved in close contact. The 2D fingerprints giving the contacts contributing most to the overall intermolecular interactions in P1–P4 are displayed in Figure 9. In all crystals, the highest intercontacts are found for H···H, and C···H/H···C interactions (

Table 6. The largest contributors to intermolecular contacts in P1–P4.

	P1	P2	P3	P4
H···H	46.9	44.0	43.2	40.0
C···H/H···C	14.6	27.5	24.7	28.8
O···H/H···O	10.0	11.1	10.0	15.2
N···H/H···N	10.8	11.5	6.7	8.7

).

This can be attributed to the significant hydrogen content of the four molecules and the fact that most of the hydrogen atoms are attached to phenyl and methyl groups which protrude out from the centers of the molecules and thus comprise a significant portion of their peripheries. Although not making the largest intermolecular contacts, the fingerprint plot for P1 is notable for the pair of sharp spikes with d_e + d_i ≈ 2.1 Å. These can be attributed to the N—H···O hydrogen bonds which, in addition to being the shortest intermolecular contact, have essentially the same H···O distance throughout the crystal. For the other three molecules, the fingerprint plots show two pairs of sharp points that extend slightly from either side of the center point. Those in P1 both have d_e + d_i ≈ 2.4 Å which is consistent with them representing the C—H···N hydrogen bonds. As the H···N contact distance is longer than the H···O distance in P1, and because it is closer in magnitude to the other intermolecular contacts in P2, these peaks are much less prominent. The same comment applies to the other two overall fingerprint plots with P3 showing peaks at d_e + d_i ≈ 2.36 and 2.20 Å and P4 having them at 2.40 and 2.36 Å. In both cases, these can be attributed to C—H···N and C—H···O hydrogen bonds, respectively.

2.4. Antiproliferative Activity

The antiproliferative activity of P1–P4 was evaluated by the MTT standard assay and sunitinib (a well-known FDA-approved multi-kinase inhibitor) as a reference against HCT 116, HepG2, and MCF-7 cell lines, and WI38 normal cell line. Compounds P3 and P4 exhibited very weak anticancer activity with IC₅₀ values above 67 µM (

Table 7. In vitro antiproliferative activity of P1–P4 against HCT 116, HepG2, and MCF-7 cancer cell lines, and a normal cell line WI38.

Compound	In Vitro Cytotoxicity IC50 (µM) *				Selectivity Index
	HCT 116	HepG2	MCF-7	WI38
P1	28.93 ± 2.1	22.70 ± 1.7	36.32 ± 2.6	85.73 ± 4.7	0.34
P2	38.68 ± 2.3	31.93 ± 2.1	40.75 ± 2.4	>100	0.37
P3	89.92 ± 4.5	88.62 ± 4.7	85.16 ± 4.4	52.49 ± 3.1	1.67
P4	92.47 ± 4.9	66.81 ± 3.8	>100	44.03 ± 2.6	1.96
Sunitinib	17.91 ± 1.3	8.38 ± 0.5	24.06 ± 2.0	55.63 ± 3.3	0.30

* Results indicate three different experiments.

and Figure 10); they show more cytotoxicity toward the normal cell line than towards the cancer cell lines. By contrast, P1 and P2 showed an antiproliferative activity comparable to the reference standard anticancer drug against the cancer cell lines as their results have less than three-fold difference compared to sunitinib (IC₅₀ values in 22.7–40.75 µM). P1 and P2 showed excellent selectivity indices as they displayed very weak cytotoxic effects on the normal cell line. The obtained results indicate that biological targets through which the tested compounds exhibit their anticancer effect could not perfectly accommodate relatively huge substitutions on position number three. Therefore, P3 and P4 exhibited weak anticancer effects.

2.5. Molecular Docking Results

Since P1–P4 compounds have no better antiproliferative activity than the reference, their binding affinity within the binding site of the human DNA topoisomerase was explored using molecular docking to better understand their modest activity. The calculated binding energies of the stable complexes P_i=1–4-DNA topoisomerase, the number of intermolecular hydrogen bonds, and the number of closest amino acids that interact with P1–P4 are summarized in

Table 8. Calculated binding energies (BE), number of hydrogen bonds (HBs), and the number of closest residues (NR) to the docked P1–P4 into DNA topoisomerase binding site.

	BE (kcal/mol)	HBs	NR 1
P1	−7.01	2	ASN A722, THR A718, DT B10, TGP C11, DA D113, DC D112
P2	−7.03	1	ARG A364, ASN A722, DT B10, TGP C11, DA D113, DC D112
P3	−7.51	1	ARG A364, ASN A722, LEU A721, DT B10, TGP C11, DA D113, DC D112
P4	−8.52	1	ARG A364, TYR A426, LYS A425, DT B10, TGP C11, DA D113, DC D112

¹ Number of amino acids of DNA Topoisomerase that interact with P1–P4.

.

P1–P4 fit well into the DNA topoisomerase binding site. They lead to the formation of stable complexes with binding energies in the range of −7 to −8.52 kcal.mol⁻¹. The negative binding energies may indicate that the inhibition of DNA topoisomerase by P1–P4 is thermodynamically favorable and spontaneous. Figure 11 and Figure 12 display, respectively, the 2D and 3D binding interaction modes established in stable P_i=1–4-DNA topoisomerase complexes. These interactions involve (i) conventional hydrogen bond interactions (ii) π-π stacked interactions, (iii) π-alkyl, (iv) π-σ, and (v-carbon hydrogen bond interactions (Figure 11 and Figure 12). Binding energies of stable P_i=1–4-DNA topoisomerase complexes vary slightly (less than 1.5 kcal-mol⁻¹) so the binding energy may not be a significant factor in explaining the observed antiproliferative activity of P1–P4 against the three cancer cell lines. Among P1–P4, P1 shows the best activity against the three cell lines and it may be that the higher activity of P1 relates to the number of hydrogen bonds it forms with the amino acids of DNA topoisomerase and the types of amino acids involved. In P2–P4, a hydrogen bond is established with ARG A364. However, in P1, its keto and amine functional groups form two hydrogen bonds with THR A178 and ASN A722 of DNA topoisomerase of distances 3.25 and 2.07 Å, respectively (Figure 11 and Figure 12). Thus, one may conclude that the higher inhibition of P1 may return to hydrogen binding established between ASN A722 and the amine of pyrazolo[3,4-d]pyrimidine.

3. Materials and Methods

3.1. Reagents and Instruments

All chemicals used in this work were purchased from commercial sources (Sigma-Aldrich, Fluka, Saint Louis, MO, USA). They were used without further purification. Mel-Temp 3.0 apparatus was used to determine the melting points. NMR spectra were generated using Bruker Advance/DPX 250 (250 MHz ¹H, 62.9MHz ¹³C) and Bruker (Billerica, MA, USA) Advance II/DPX 400 (400 MHz ¹H, 100 MHz ¹³C) spectrometers.

3.2. Synthesis

Pyrazolo [3,4-d] pyrimidine derivatives (P1–P4) were synthesized by following the procedure mentioned under Scheme 1. Based on the literature, 2-(1-ethoxyethylidene)malononitrile (A) and phenyl hydrazine were refluxed in ethanol for 2 h to afford 5-amino-3-methyl-1-phenyl-1H-pyrazole-4-carbonitrile (B) [20]. The latter was reacted with formic acid under reflux for 7 h to give compound P1 in good yield, which was reacted with the selected alkylating agent: methyl iodide, propargyl bromide, phenacyl bromide to give the final products P2, P3, and P4, respectively, in good yield.

3.2.1. Synthesis of P1

A solution of 5-amino-3-methyl-1-phenyl-1H-pyrazole-4-carbonitrile (B) (1.0 g, 5.04 mmol) in formic acid (30 mL) was refluxed for 7 h. The final mixture was poured into ice water and the resulting precipitate was filtered off, dried, and recrystallized from ethanol (Scheme 1).

Yield: 83%. mp: 153–155 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.34 (s, 1H), 8.13 (s, 1H), 8.03–8.01 (m, 2H), 7.54–7.50 (m, 2H), 7.37–7.33 (m, 1H), 2.52 (s, 3H) (Figure S5).¹³C NMR (101 MHz, DMSO-d₆) δ 157.99, 152.24, 148.88, 145.93, 138.29, 129.11, 126.62, 121.36, 105.74, 13.37 (Figure S6).

3.2.2. Synthesis of P2–P4

A DMF (5 mL) solution containing P1 (0.1 g, 0.44 mmol), K₂CO₃ (1.2 equiv.), and tetra n-butylammonium bromide (TBAB) catalyst was stirred for 10 min. Then, the selected alkylating agent (1.2 equiv.) was added to the solution, and the mixture was stirred overnight. The mixture was then poured into ice water and the resulting precipitate was filtered off, dried, and recrystallized from ethanol yielding the final product (Scheme 1).

3,5-Dimethyl-1-phenyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (P2)

Yield: 70%. mp: 143–145 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.40 (s, 1H), 8.02–7.99 (m, 2H), 7.54–7.50 (m, 2H), 7.37–7.33 (m, 1H), 3.47 (s, 3H), 2.52 (s, 3H) (Figure S7).¹³C NMR (101 MHz, DMSO-d₆) δ 157.60, 151.90, 151.70, 145.86, 138.27, 129.24, 126.66, 121.20, 104.94, 33.15, 13.41 (Figure S8).

3-Methyl-1-phenyl-5-(prop-2-yn-1-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (P3)

Yield 65%, mp = 155–157 °C. ¹H NMR (250 MHz, DMSO-d₆) δ 8.54 (s, 1H), 8.02–7.99 (m, 2H), 7.54 (m, 2H), 7.39 (m, 1H), 4.82 (d, J = 2.5 Hz, 2H), 3.43 (t, J = 2.5 Hz, 1H), 2.55 (s, 3H) (Figure S9).¹³C NMR (63 MHz, DMSO-d₆) δ 156.31, 151.44, 150.71, 146.10, 138.02, 129.21, 126.87, 121.47, 104.84, 78.65, 75.75, 34.63, 13.34 (Figure S10).

3-Methyl-5-(2-oxo-2-phenylethyl)-1-phenyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (P4)

Yield 69%, mp = 1170–172 °C. ¹H NMR (250 MHz, DMSO-d₆) δ 8.43 (s, 1H), 8.12–8.03 (m, 4H), 7.75–7.73 (m, 1H), 7.65–7.53 (m, 4H), 7.42–7.35 (m, 1H), 5.64 (s, 2H), 2.53 (s, 3H) (Figure S11).¹³C NMR (63 MHz, DMSO-d₆) δ 192.84, 156.91, 151.84, 151.71, 146.07, 138.12, 134.27, 129.23, 129.07, 128.10, 126.82, 121.44, 104.85, 51.48, 13.32 (Figure S12).

The possible tautomeric forms of P1 (P1–P1″) are displayed in Scheme 2. It possesses three reactive sites towards the alkylating agents N5, N7, and O of OH at C4. The alkylation reactions carried out under liquid–solid phase transfer catalysis lead in each case to only one alkylated product, P2–P4, showing that N5 of the pyrazolopyrimidine is the most reactive site of the heterocyclic compound P1.

3.3. Single Crystal X-Ray Diffraction

The crystal structures of P1–P4 were generated from X-ray intensity data collected on a Bruker D8 QUEST PHOTON 3 diffractometer. More details on the X-ray diffraction methodology may be found in our previous studies [21].

3.4. Hirshfeld Surface Study

Hirshfeld surfaces and fingerprint plots of P1–P4 were generated with Crystal Explorer 3.0 [22,23]. d_norm was mapped from −0.60 a.u (blue) to 1.25 a.u (red), while 2D fingerprints were plotted and displayed on a 0.8–2.4 Å expanded scale.

3.5. DFT Calculations

The stable geometries of P1–P4 were optimized in solvent using the B3LYP hybrid functional along with the basis set 6–311++G(d,p). The frequency calculations confirm that the optimized geometries of P1–P4 are true minima. The experimental atom coordinates of P1–P4 were used as starting inputs for the optimization. The solvent was modulated using the implicit polarizable continuum model (PCM) and chloroform as solvent [24]. Theoretical calculations were accomplished using the Gaussian 16 software [25].

3.6. Molecular Docking Study

The binding affinities of P1–P4 into the human DNA topoisomerase binding site as the target of anticancer agents were explored with the Autodock package [26]. The structural geometries of the human topoisomerase I-DNA and its EHD ligand were downloaded from the data website RCSB (PDB file 1T8I) [27]. The re-docking of EHD is relatively well reproduced, with an RMSD value of 0.30 Å and binding energy of −10.50 kcal-mol⁻¹. Details of the docking calculations may be found in our previous study [21].

3.7. Cytotoxicity Assay

The antiproliferative activity of P1–P4 was assessed by using an MTT standard assay and sunitinib drug towards HCT 116, HepG2, MCF-7 cells, and WI38 normal cell line using the previously described methodology [21].

4. Summary

Four new pyrazolo[3,4-d]pyrimidines (P1–P4) have been synthesized and their structures determined using NMR spectroscopy and single crystal X-ray diffraction. Hirshfeld surfaces and 2D fingerprints revealed that H···H and C···H/H···C are the major intermolecular contacts in P1–P4. Bond lengths, bond angles, and torsion angles of P1–P4 are relatively well reproduced at the B3LYP level of theory with correlation coefficients higher than 90%. The antiproliferative activity of P1–P4 is weak compared to the antiproliferative activity of the reference standard anticancer drug against the cancer cells with IC₅₀ of 22.7–40.75 µM. Molecular docking reveals that DNA topoisomerase inhibition by P1–P4 is a spontaneous, thermodynamically favorable process and is influenced by the number of hydrogen bonds and the types of amino acids involved in the formation of hydrogen bonds between P1–P4 and the amino acids of DNA topoisomerase.