In vivo evaluation of novel synthetic pyrazolones as CDK9 inhibitors with enhanced pharmacokinetic properties

Abstract

Aim: The structural optimization of our recently reported CDK9 inhibitor to furnish novel aminopyrazolones and methylpyrazolones with improved pharmacokinetics.

Materials & methods: The synthesis of the targeted compounds was accomplished via conventional, grinding and microwave-assisted processes. The cytotoxicity of them was assayed against three carcinomas.

Results: Analogs 2, 4 and 6 showed significant cytotoxicity and selectivity toward all tested cells. They also displayed potent CDK9 inhibition. Compound 6 arrested MCF-7 cycle at G2/M phase by stimulating the apoptotic pathway. The in vivo biodistribution of radiolabeled compound 6 displayed a potent targeting capability of ¹³¹I in solid tumors.

Conclusion: Entity 6 is a potent CDK9 inhibitor where ¹³¹I-compound 6 can be used as a significant radiopharmaceutical imaging tool for tumors.

Graphical Abstract

Plain language summary

Article highlights.

• Structural optimization of diaminopyrazole to furnish novel aminopyrazolones and methylpyrazolones with improved pharmacokinetic properties.

• Three compounds 2, 4 and 6 showed significant cytotoxic activity and selectivity toward all tested cancerous cells.

• Compounds 2, 4 and 6 also displayed potent CDK9 inhibition with an IC₅₀ range equal to 0.496–7.149 μM.

• The most effective CDK9 inhibitor 6 arrested the cell cycle of MCF-7 at the G2/M phase by stimulating the apoptotic pathway.

• In vivo biodistribution study of ¹³¹I-6 displayed a potent targeting ability of ¹³¹I in solid tumor suggesting that ¹³¹I-6 might be used as a promising radiopharmaceutical imaging agent for cancer.

1. Background

The inhibition of cyclin-dependent kinases (CDKs) has been documented as an effective treatment strategy to manage cancer. Palbociclib, ribociclib and abemaciclib are examples of CDK inhibitors that are currently under investigation, either as monotherapy or as co-therapy with aromatase inhibitors. Palbociclib and ribociclib have already been confirmed through the US FDA as combinatorial medications for the management of metastatic breast cancer in postmenopausal women. CDK9 is one member of this family of kinases that is crucial to cellular and gene transcription. It also participates in programmed cellular death and has long been considered a medicinal target in cancer therapy [1]. Several synthetic molecules have been developed as inhibitors of protein kinases, including CDK9, in recent years (Figure 1) [2–5]. Among these, dinaciclib (I), seliciclib (II) and alvocidib (III) induced the apoptotic cell death of human colorectal, prostate, lymphocytic leukemia and breast cancers [1,6,7].

Figure 1.

Upper panel: CDK9 inhibitors in clinical trials; lower panel: aminopyrazole and sulfaguanidine-incorporated anticancer agents.

From one point of view, pyrazole is a promiscuous scaffold targeting multiple types of protein kinases [8]. It has been considered the major concern of cancer therapy research and emerged as significant core in diverse protein kinases inhibitors such as JNK3 [9], VEGFR-2 [10,11], EGFR^WT [10–12], EGFR^T790M [12], B-RAF [10], PI3K [13], pan-FGFR [14], ERK and RIPK3 kinases [15]. Aminopyrazoles have a long history of inclusion in the skeleton of various bioactive molecules especially chemotherapeutics with CDK inhibition potential (Figure 1) [16–23]. The 4-aminopyrazole IV was reported as a powerful antitumor drug in in vivo models for human colorectal and ovarian carcinomas [16]. Additionally, aminopyrazole-indole hybrids V and VI have demonstrated potent in vitro cytotoxicity toward the HepG2 cell line compared with doxorubicin. Enzyme assays have shown that both compounds showed superior CDK inhibition effect compared with roscovitine [17]. From another point of view, sulphaguanidine derivatives have gained significant attention in the last years depending on their pharmacological importance, especially as anticancer agents [24–27]. For example, the acridine-linked sulfaguanidine VII has demonstrated more than 50% suppression of several carbonic anhydrases efficacy [26]. Additionally, the quinazoline-linked sulphaguanidine VIII has shown a considerable antiproliferative effect toward lung and breast carcinomas [24].

1.1. Rationale & aim of the work

Two known CDK9 inhibitors in the Protein Data Bank (PDB) were realized to be derived from 3,5-diaminopyrazole (CAN-508, IX) [28], and the N-morpholinosulfonylphenylaminopyrimidine X [18,29]. In our most recent report, we considered the structural features of both inhibitors to identify compounds XI and XII as potent cytotoxic agents for HepG2, HCT116, and MCF-7 tumor cells. Both compounds have also shown a powerful effect as CDK9 inhibitors with IC₅₀ range equal to 0.16–1.69 μM [30]. Molecular docking experiments of both compounds revealed the significance of one amino functionality out of two of the 3,5-diaminoazopyrazole XI and one carbonyl group out of two in the pyrazolidine-3,5-dione XII for an effective hydrogen bonding (HB) interaction with CDK9 (Figure 2) [30].

Figure 2.

Rationalized design of new CDK9 inhibitors with improved pharmacokinetic properties.

As a key element, HB is implicated in numerous physicochemical phenomena like DNA base-pairing and stability of the 3D structures of drug-target complexes [31,32]. The formation of such an intermolecular connectivity force necessitates the presence of both hydrogen bond donor (HBD) and hydrogen bond acceptor groups [33,34]. However, HBD groups have been documented as causative agents for more difficulties in drug design than hydrogen bond acceptors [32]. HB affects the pharmacokinetic profiles (absorption, distribution, metabolism, excretion, and toxicity; ADMET) of medicaments. Consequently, the permeability and in vivo biodistribution of this drug could be affected. Recently, the exclusion of nonessential HBD groups has become a significant tactic in medicinal chemistry, for example, in the design of nirmatrelvir SARS-CoV-2 M^pro inhibitor candidate [35].

Considering these articulated scientific facts on the exclusion of nonessential HBD groups as a significant tactic in medicinal chemistry, and as an extension to our previous article [30], we report here the structural optimization of the last reported series of sulfaguanidine-linked 3,5-diaminoazopyrazoles and azopyrazolidine-3,5-diones. This structural optimization was aimed toward the identification of new potent CDK9 inhibitors with improved pharmacokinetic properties containing aminopyrazolone and methylpyrazolone scaffolds. Moreover, biodistribution studies provide a possible approach for estimating the newly synthesized analogs [36]. Radiolabeling these substances with appropriate radionuclides is the most applicable strategy for biodistribution analysis. The radioactive uptake of tissues and/or organs is computed at various periods [37]. These carriers must be radiolabeled under appropriate circumstances that result in minimal or negligible structural alterations to the carrier [38].

2. Materials & methods

2.1. Conventional synthesis

The specifications of devices utilized for the characterization of novel compounds were attached in the Supplementary materials. The progression of reactions was observed through thin-layer chromatography (TLC) with ethylacetate: hexane (3:1) as eluent. The synthesis of compounds 1–3, 7–9 was accomplished via conventional method only following the reported procedure [39].

2.1.1. Formation of Ethyl-2-cyano-2-{[4-(N-(diaminomethylene)sulfamoyl)phenyl]diazinyl}acetate (1)

An equal volume of concentrated HCl (5 ml) and water (5 ml) was mixed and used to dissolve sulphaguanidine (2.14 g, 0.01 mol). The mixture was cooled to 5–10°C in an ice bath before adding a cold aqueous sodium nitrite solution (0.69 g, 0.01 mol) while stirring. The formed diazonium salt was filtered into a cold sodium acetate solution (4 g), and then ethyl cyanoacetate (1.13 g, 1.06 ml, 0.01 mol) in ethanol (25 ml) was added under stirring for 1 h. The resulting precipitate was recrystallized twice from ethanol to afford compound 1.

Pale yellow crystals, m.p. 226–228°C. IR (cm^-1) υ: 3412, 3321, 3233, 3191 (NH₂), 2226 (CN), 1695 (C=O), 1626 (C=N), 1595 (N=N). ¹H NMR (DMSO-d₆) δ (ppm): 1.19 (t, 3H, J = 6.3 Hz, CH₂CH₃), 2.07 (s, 1H, N=N-CH), 4.10 (q, 2H, J = 6.3 Hz, CH₂CH₃), 6.66 (s, 4H, 2NH₂, D₂O exchangeable), 7.28 (d, 2H, J = 8.6 Hz, C₆H₄-C_2,6-H), 7.56 (d, 2H, J = 8.6 Hz, C₆H₄-C_3,5-H). ¹³C NMR (DMSO-d₆) δ (ppm): 15.9 (CH₃), 58.5 (CH), 62.5 (CH₂), 110.0 (CN), 119.1, 126.8, 131.7, 154.0, 158.4, 178.5 (C=O). MS (m/z): 338 (M⁺, 29.28%). Anal. Calcd for C₁₂H₁₄N₆O₄S (338): C, 42.60; H, 4.17; N, 24.84; S, 9.48%. Found: C, 42.57; H, 4.28; N, 24.77; S, 9.32%.

2.1.2. General procedure for preparation of compounds (2–6)

A solution of analog 1 (3.38 g, 0.01 mol) in ethanol (30 ml) was heated under reflux for a period of 4–8 h with each of hydrazine hydrate (0.5 g, 0.5 ml, 0.01 mol), phenylhydrazine (1.08 g, 0.98 ml, 0.01 mol), semicarbazide hydrochloride (1.12 g, 0.01 mol), thiosemicarbazide (0.91 g, 0.01 mol) or benzohydrazide (1.36 g, 0.01 mol). Compounds 2–6 were obtained, respectively, by filtering the formed solids after cooling and recrystallizing them with the appropriate solvent.

2.1.2.1. 4-[(3-Amino-5-oxo-4,5-dihydro-1H-pyrazol-4-yl)diazinyl]-N-(diaminomethylene)benzenesulfonamide (2)

Gray crystal, recrystallized from methanol, m.p. 212–214°C. IR (cm^-1) υ: 3452, 3342, 3266, 3216 (NH₂ & NH), 1684 (C=O), 1630, 1572 (C=N), 1529 (N=N). ¹H NMR (DMSO-d₆) δ (ppm): 2.07 (s, 1H, N=N-CH), 6.66 (s, 4H, 2NH₂, D₂O exchangeable), 7.28 (d, 2H, J = 8.6 Hz, C₆H₄-C_2,6-H), 7.56 (d, 2H, J = 8.6 Hz, C₆H₄-C_3,5-H), 8.85 (s, 1H, NH, D₂O exchangeable), 9.65 (s, 2H, NH₂, D₂O exchangeable). ¹³C NMR (DMSO-d₆) δ (ppm): 62.5 (CH), 116.4 (2C), 126.6 (2C), 131.6, 147.0, 162.0, 168.4, 207.0 (C=O). MS (m/z): 324 (M⁺, 25.47%). Anal. Calcd for C₁₀H₁₂N₈O₃S (324): C, 37.03; H, 3.73; N, 34.55; S, 9.89%. Found: C, 36.91; H, 3.75; N, 34.48; S, 10.02%.

2.1.2.2. 4-((3-Amino-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl)diazenyl)-N-(diaminomethylene)benzenesulfonamide (3)

Orange powder, recrystallized from methanol, m.p. 220–221°C. IR (cm^-1) υ: 3431, 3331, 3181, 3162 (NH₂), 1661 (C=O), 1624, 1592 (C=N), 1555 (N=N). ¹H NMR (DMSO-d₆) δ (ppm): 2.07 (s, 1H, N=N-CH), 6.48 (s, 2H, NH₂, D₂O exchangeable), 6.69 (s, 4H, 2NH₂, D₂O exchangeable), 7.12 (t, 1H, J = 7.6 Hz, C₆H₅-C₄-H); 7.12 (t, 2H, J = 7.6 Hz, C₆H₅-C_3,5-H); 7.70–7.76 (m, 4H, C₆H₅-C_2,6-H & C₆H₄-C_2,6-H); 7.91 (d, 2H, J = 8.4 Hz, C₆H₄-C_3,5-H). ¹³C NMR (DMSO-d₆) δ (ppm): 89.5 (CH), 115.8 (2C), 117.6 (2C), 127.5 (2C), 129.3 (2C), 131.2, 140.1, 142.5, 155.9, 159.5, 168.5; 181.5 (C=O). MS (m/z): 400 (M⁺, 12.25%). Anal. Calcd for C₁₆H₁₆N₈O₃S (400): C, 47.99; H, 4.03; N, 27.98; S, 8.01%. Found: C, 48.08; H, 3.88; N, 27.94; S, 8.09%.

2.1.2.3. 3-Amino-4-{[4-(N-(diaminomethylene)sulfamoyl)phenyl]diazenyl}-5-oxo-4,5-dihydro-1H-pyrazole-1-carboxamide (4)

Yellow powder, recrystallized from methanol, m.p. 250–252°C. IR (cm^-1) υ: 3434, 3345, 3224 (NH₂), 1745, 1661 (C=O), 1618 (C=N), 1554 (N=N). ¹H NMR (DMSO-d₆) δ (ppm): 1.91 (s, 1H, N=N-CH), 4.13 (s, 2H, pyrazole-NH₂, D₂O exchangeable), 5.89 (s, 2H, CONH₂, D₂O exchangeable), 6.71 (s, 4H, 2NH₂, D₂O exchangeable), 7.32 (d, 2H, J = 7.4 Hz, C₆H₄-C_2,6-H), 7.61 (d, 2H, J = 7.4 Hz, C₆H₄-C_3,5-H). ¹³C NMR (DMSO-d₆) δ (ppm): 97.8 (CH), 118.2, 119.0, 126.9, 129.1 (2C), 138.5, 156.8, 158.5, 166.0 (CONH₂), 172.6 (C=O). MS (m/z): 367 (M⁺, 14.36%). Anal. Calcd for C₁₁H₁₃N₉O₄S (367): C, 35.97; H, 3.57; N, 34.32; S, 8.73%. Found: C, 36.04; H, 3.55; N, 34.19; S, 8.70%.

2.1.2.4. 3-Amino-4-{[4-(N-(diaminomethylene)sulfamoyl)phenyl]diazenyl}-5-oxo-4,5-dihydro-1H-pyrazole-1-carbothioamide (5)

Yellow powder, recrystallized from acetone, m.p. 278–280°C. IR (cm^-1) υ: 3495, 3437, 3344, 3226 (NH₂), 1746 (C=O), 1617 (C=N), 1553 (N=N). ¹H NMR (DMSO-d₆) δ (ppm): 1.90 (s, 1H, N=N-CH), 6.74 (s, 4H, 2NH₂, D₂O exchangeable), 7.21 (s, 2H, pyrazole-NH₂, D₂O exchangeable), 7.53 (d, 2H, J = 8.6 Hz, C₆H₄-C_2,6-H), 7.77 (d, 2H, J = 8.6 Hz, C₆H₄-C_3,5-H), 8.63 (s, 2H, CSNH₂, D₂O exchangeable). ¹³C NMR (DMSO-d₆) δ (ppm): 96.5 (CH), 127.6, 127.7, 140.6, 141.4, 143.6, 145.3, 158.5, 160.7, 161.4 (C=O), 181.6 (C=S). MS (m/z): 383 (M⁺, 10.02%). Anal. Calcd for C₁₁H₁₃N₉O₃S₂ (383): C, 34.46; H, 3.42; N, 32.88; S, 16.72%. Found: C, 34.50; H, 3.41; N, 32.78; S, 16.74%.

2.1.2.5. 4-[(3-Amino-1-benzoyl-5-oxo-4,5-dihydro-1H-pyrazol-4-yl)diazinyl]-N-(diaminomethylene)benzenesulfonamide (6)

Yellow powder, recrystallized from acetone, m.p. 284–286°C. IR (cm^-1) υ: 3412, 3321, 3231, 3191 (NH₂), 1695, 1659 (C=O), 1622, 1598 (C=N), 1536 (N=N). ¹H NMR (DMSO-d₆) δ (ppm): 1.91 (s, 1H, N=N-CH), 6.70 (s, 4H, 2NH₂, D₂O exchangeable), 7.45–7.83 (m, 9H, Ar-H), 9.80 (s, 2H, pyrazole-NH₂, D₂O exchangeable). ¹³C NMR (DMSO-d₆) δ (ppm): 91.51 (CH), 127.3, 127.4, 127.7, 127.9, 128.8, 131.6, 133.8, 139.7, 141.5, 143.7, 150.2, 158.5, 160.7, 163.4, 166.4 (C=O), 172.5 (pyrazole-C=O). MS (m/z): 428 (M⁺, 12.58%). Anal. Calcd for C₁₇H₁₆N₈O₄S (428): C, 47.66; H, 3.76; N, 26.16; S, 7.48%. Found: C, 47.71; H, 3.69; N, 26.21; S, 7.51%.

2.1.3. Ethyl 2-{[4-(N-(diaminomethylene)sulfamoyl)phenyl]diazenyl}-3-oxobutanoate (7)

Compound 7 was prepared using the same method in synthesizing compound 1 where the replacement of ethyl cyanoacetate with ethyl acetoacetate (1.30 g, 1.27 ml, 0.01 mol). The resulting precipitate was recrystallized twice from methanol to give 7.

Yellow powder, recrystallized from acetone, m.p. 181–182°C. IR (cm^-1) υ: 3449, 3336, 3230 (NH₂), 1690, 1684 (C=O), 1630 (C=N), 1527 (N=N). ¹H NMR (DMSO-d₆) δ (ppm): 1.25 (t, 3H, J = 6.3 Hz, CH₂CH₃), 2.07 (s, 3H, CH₃), 4.25 (q, 2H, J = 6.3 Hz, CH₂CH₃), 4.38 (s, 1H, N=N-CH), 6.66 (s, 4H, 2NH₂, D₂O exchangeable), 7.44 (d, 2H, J = 8 Hz, C₆H₄-C_2,6-H), 7.68 (d, 2H, J = 8 Hz, C₆H₄-C_3,5-H). ¹³C NMR (DMSO-d₆) δ (ppm): 15.5 (CH₂CH₃), 25.9 (CH₃), 62.5 (CH₂CH₃), 73.7 (CH), 123.7, 127.6, 143.4, 154.4, 158.5, 175.5 (C=O); 197.6 (COO). MS (m/z): 355 (M⁺, 16.54%). Anal. Calcd for C₁₃H₁₇N₅O₅S (355): C, 43.94; H, 4.82; N, 19.71; S, 9.02%. Found: C, 44.08; H, 4.66; N, 19.84; S, 8.89%.

2.1.4. General method for preparation of analogs (8-12)

A solution of compound 7 (3.55 g, 0.01 mol) in ethanol (30 ml) was refluxed for 4–12 h with each of hydrazine hydrate (0.5 g, 0.5 ml, 0.01 mol), phenyl hydrazine (1.08 g, 0.98 ml, 0.01 mol), semicarbazide hydrochloride (1.12 g, 0.01 mol), thiosemicarbazide (0.91 g, 0.01 mol) or benzohydrazide (1.36 g, 0.01 mol). The solid that was obtained upon cooling was gathered by filtration and then recrystallized with proper solvent to produce compounds 8–12, respectively.

2.1.4.1. N-(Diaminomethylene)-4-[(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-4-yl)diazinyl]benzenesulfonamide (8)

Orange powder, recrystallized from methanol, m.p. 259–261°C. IR (cm^-1) υ: 3448, 3414, 3309, 3201 (NH₂ & NH), 1673 (C=O), 1639, 1589 (C=N), 1544 (N=N). ¹H NMR (DMSO-d₆) δ (ppm): 2.07 (s, 3H, CH₃), 2.14 (s, 1H, N=N-CH), 6.69 (s, 4H, 2NH₂, D₂O exchangeable), 7.59 (d, 2H, J = 8.8 Hz, C₆H₄-C_2,6-H), 7.75 (d, 2H, J = 8.8 Hz, C₆H₄-C_3,5-H), 11.59 (s, 1H, NH, D₂O exchangeable). ¹³C NMR (DMSO-d₆) δ (ppm): 25.9 (CH₃), 62.5 (CH), 114.7, 115.8, 122.5, 127.7, 146.9, 157.9, 187.4 (C=O). MS (m/z): 323 (M⁺, 8.14%). Anal. Calcd for C₁₁H₁₃N₇O₃S (323): C, 40.86; H, 4.05; N, 30.32; S, 9.92%. Found: C, 40.91; H, 3.99; N, 30.28; S, 9.99%.

2.1.4.2. N-(Diaminomethylene)-4-[(3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl)diazinyl]benzenesulfonamide (9)

Orange powder, recrystallized from methanol, m.p. 229–231°C. IR (cm^-1) υ: 3429, 3341, 3191 (NH₂), 1657 (C=O), 1633, 1590 (C=N), 1546 (N=N). ¹H NMR (DMSO-d₆) δ (ppm): 2.04 (s, 3H, CH₃), 2.25 (s, 1H, N=N-CH), 7.20 (s, 4H, 2NH₂, D₂O exchangeable), 7.42–7.79 (m, 9H, Ar-H). ¹³C NMR (DMSO-d₆) δ (ppm): 22.1 (CH₃), 66.8 (CH), 116.5, 118.3, 125.4, 127.7, 129.5 (2C), 129.6 (2C), 138.3, 141.4, 144.0, 149.2, 156.8, 158.6, 178.6 (C=O). MS (m/z): 400 (M+1, 15.26%). Anal. Calcd for C₁₇H₁₇N₇O₃S (399): C, 51.12; H, 4.29; N, 24.55; S, 8.03%. Found: C, 51.04; H, 4.28; N, 24.61; S, 7.99%.

2.1.4.3. 4-{[4-(N-(Diaminomethylene)sulfamoyl)phenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazole-1-carboxamide (10)

Yellow powder, recrystallized from methanol, m.p. 248–250°C. IR (cm^-1) υ: 3445, 3403, 3341, 3208 (NH₂), 1698 (C=O), 1630, 1591 (C=N), 1541 (N=N). ¹H NMR (DMSO-d₆) δ (ppm): 2.06 (s, 3H, CH₃), 2.22 (s, 1H, N=N-CH), 6.78 (s, 4H, 2NH₂, D₂O exchangeable), 7.14 (s, 2H, CONH₂, D₂O exchangeable), 7.26–7.39 (m, 4H, Ar-H). ¹³C NMR (DMSO-d₆) δ (ppm): 17.3(CH₃), 62.5 (CH), 127.6, 127.7, 145.3, 145.9, 151.1, 157.8, 158.6, 166.7 (2C=O). MS (m/z): 366 (M⁺, 7.29%). Anal. Calcd for C₁₂H₁₄N₈O₄S (366): C, 39.34; H, 3.85; N, 30.59; S, 8.75%. Found: C, 39.28; H, 3.89; N, 30.51; S, 8.88%.

2.1.4.4. 4-{[4-(N-(Diaminomethylene)sulfamoyl)phenyl]diazenyl}-3-methyl-5-oxo-4,5-dihydro-1H-pyrazole-1-carbothioamide (11)

Orange powder, recrystallized from methanol, m.p. 268–270°C. IR (cm^-1) υ: 3422, 3363, 3284, 3249, 3178, 3119 (NH₂), 1677 (C=O), 1632, 1599 (C=N), 1552 (N=N). ¹H NMR (DMSO-d₆) δ (ppm): 2.14 (s, 3H, CH₃), 2.25 (s, 1H, N=N-CH), 6.70 (s, 4H, 2NH₂, D₂O exchangeable), 7.46–7.79 (m, 4H, Ar-H), 9.44 (s, 2H, CSNH₂, D₂O exchangeable). ¹³C NMR (DMSO-d₆) δ (ppm): 25.9 (CH₃), 66.8 (CH), 116.9, 127.7, 128.2, 141.8, 143.9, 158.5, 171.8 (C=O), 207.0 (C=S). MS (m/z): 382 (M⁺, 12.28%). Anal. Calcd for C₁₂H₁₄N₈O₃S₂ (382): C, 37.69; H, 3.69; N, 29.30; S, 16.77%. Found: C, 37.52; H, 3.69; N, 29.37; S, 16.77%.

2.1.4.5. 4-[(1-Benzoyl-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-4-yl)diazinyl]-N-(diaminomethylene)benzenesulfonamide (12)

Yellow crystal, recrystallized from methanol, m.p. 284–286°C. IR (cm^-1) υ: 3448, 3363, 3324, 3214 (NH₂), 1721, 1668 (C=O), 1631 (C=N), 1543 (N=N). ¹H NMR (DMSO-d₆) δ (ppm): 2.07 (s, 3H, CH₃), 2.25 (s, 1H, N=N-CH), 6.69 (s, 4H, 2NH₂, D₂O exchangeable), 7.47–7.92 (m, 9H, Ar-H). ¹³C NMR (DMSO-d₆) δ (ppm): 22.1 (CH₃), 66.8 (CH), 115.8, 127.7, 127.9, 128.4, 129.0 (2C), 130.0, 142.1, 155.8, 158.5, 172.1, 178.6 (2C=O). MS (m/z): 427 (M⁺, 7.68%). Anal. Calcd for C₁₈H₁₇N₇O₄S (427): C, 50.58; H, 4.01; N, 22.94; S, 7.50%. Found: C, 50.50; H, 4.07; N, 23.01; S, 7.39%.

2.2. Microwave & grinding synthesis

In the microwave and grinding techniques, the same reactant amounts as in the conventional reactions were used without solvent. TLC was used to monitor the completion of the reaction. The solids formed were washed with methanol three-times and recrystallized from the proper solvent. Microwave irradiation techniques were carried out in Anton Paar monowave 300 using “10 ml” borosilicate glass vials for 1–6 min. Grinding reactions were carried in a porcelain mortar with a pestle for 8–25 min. The obtained product of the same reaction by using the three techniques was identical in m.p., mixed m.p. and TLC. The microwave reactions gave the highest yields with the lowest times than grinding where the conventional method came in the last.

2.3. Biological estimation

2.3.1. Antiproliferative assay

All the prepared compounds were assayed for their antiproliferative effect against three cancerous cells through the MTT technique utilizing doxorubicin as the control. The cells were treated with the synthesized analogs at concentrations 100, 50, 25, 12.5, 6.25, 3.125 and 1.56 μM and incubated for 24 h. The absorbance was detected at a wavelength of 570 nm utilizing a plate reader (EXL 800 USA) according to the reported method [40].

2.3.2. CDK9 inhibition

Estimation of CDK9 inhibition was accomplished using Biosciences CDK9 Kit (San Diego, USA) according to the manufacturer’s prescript. The experiment was carried out three-times, and IC_50s were calculated following the previously reported procedure [41].

2.3.3. Cellular apoptosis assay

The apoptosis detection for aminopyrazolone 6 was applied employing an Annexin-V-FITC/PI kit and FACS Caliber flow cytometer guided by the reported method [42].

2.3.4. Cell cycle analysis

The influence of entity 6 on the MCF-7 cell cycle was determined through a FACS Caliber flow cytometer referring to the reported technique [43].

2.4. In silico studies

2.4.1. Molecular docking study

MOE software was used in molecular docking where the crystal structure of CDK9 (CAN-508) was attained from the Protein Data Bank (PDB ID: 3TNH; Resolution: 3.20 Å; https://www.rcsb.org/structure/3TNH) [28]. The crystal structure was adjusted, the energy was minimized, then a docking procedure was carried out for all structures and the findings were resolved following the reported method [44].

2.5. Biodistribution study

Compounds 6 and XI were radiolabeled with ¹³¹I to estimate their biodistribution.

2.5.1. Radiolabeling technique

Both compounds underwent radioiodination by the use of oxidizing agents, specifically chloramine-T guided by the attached method in Supplementary materials. The following equation was applied to calculate percent RE.

$$\% \text{RE}=\frac{\mathrm{Radioactivity of radiolabeled compound}}{\mathrm{Total activity}}\times 100$$

2.5.2. In vivo evaluation of the prepared radiolabeled compounds

2.5.2.1. Induction of tumor in mice

Ehrlich-Ascites Carcinoma was used in solid tumor modeling in mice. A 7-day-old female downer Swiss Albino mouse was used to extract 0.1 ml of the cell line, which was then mixed with saline to yield 12.5 × 10⁶ cells/ml. The right thigh was injected intramuscularly with the prior mixture (0.2 ml). The mice were housed in cages protected from metabolic lead. The growth of the tumor became visible after 10–15 days, following the previously reported standard procedure [45], which is detailed in the Supplementary materials.

2.5.2.2. Biodistribution study of ¹³¹I-compound 6 & ¹³¹I-compound XI in normal & tumor-induced mice

The biological behavior of radiolabeled compounds 6 and XI in vivo was studied, applying the previously reported protocol [46,47], as detailed in the Supplementary file. Every procedure involving animals was conducted according to the guidelines approved by the Ethics Committee for Experimental Studies (Human& Animal subject) at the National Center for Research Radiation and Technology-Egyptian Atomic Energy Authority, Cairo, Egypt (9A/24). Each radiolabeled chemical was given intravenously via the mouse's tail vein in an amount equal to 3.7 MBq in 100 μl. Thiopental was used as an anesthetic for the dissection of mice at 0.5, 1, 2 and 4 h after injection.

3. Results & discussion

3.1. Chemistry

For the synthesis of heterocyclic compounds, microwave and grinding procedures were employed by the principles of green chemistry [30,48,49]. Diazonium salts were considered an active electrophilic reagent [50], which was utilized in the synthesis of a variety of heterocycles through coupling with different active methylene-bearing derivatives in a basic solution [51]. Hence, diazotization of sulphaguanidine with sodium nitrite in the presence of c. HCl was pursued via coupling with ethyl cyanoacetate and ethyl acetoacetate in ethanol/sodium acetate solution to yield the starting compounds 1 and 7, respectively (Figure 3). Such starting compounds were used as precursors to synthesize the target aminopyrazolone and methylpyrazolone derivatives. The construction of starting compounds was confirmed via their spectral information. Infrared charts of 1 and 7 presented ester carbonyl functionality at the absorption range of 1695–1690 cm^-1 in addition to cyano group absorption in analog 1 and ketonic carbonyl functionality absorption of compound 7 at 2226 and 1684 cm^-1, respectively. ¹H NMR spectra of 1 and 7 presented triplet and quartet of ester ethyl carboxylate moiety at δ 1.19 -1.25 and 4.10 - 4.25 ppm, respectively. The existence of a singlet in ¹H NMR of 7 at δ 2.07 ppm assigned for ketonic CH₃ protons supported the designed structure.

Figure 3.

Upper panel: synthesis of the starting compounds 1 and 7, Lower panel: synthesis of aminopyrazolone derivatives 2-6.

The condensation of nitrile [52,53] or carbonyl [53–55] bearing scaffolds with hydrazines was reported to be carried out by simple refluxing in ethanol. Herein, aminopyazolone derivatives 2–6 were constructed through the cyclocondensation of ethyl cyanoacetate analog 1 with hydrazine hydrate, phenylhydrazines, semicarbazide, thiosemicarbazide and benzohydrazide (Figure 3). The mechanistic pathway for this reaction was offered to progress by the nucleophilic addition of one of the hydrazine NH₂ groups to the electrophilic ethyl carboxylate carbonyl function followed by proton exchange and removal of ethanol moiety to give the nonisolated acid hydrazide. A concomitant nucleophilic addition of the second NH₂ functionality to nitrile moiety pursued by cyclization and aromatization resulted in the formation of aminopyrazolones 2–6 (Figure 4). The structures of aminopyrazolones 2–6 were emphasized via the absence of absorption bands for cyano function in the IR chart, together with the lack of triplet and quartet signals of ethyl carboxylate moiety in ¹H NMR spectra. Besides, D₂O exchangeable singlets were remarked at the range of 4.13–9.80 ppm, attributed to the pyrazole amino group. As well, the manifestation of D₂O exchangeable singlets at 5.89 and 8.63 ppm in 4 and 5 corresponds to carboxamide and carbothioamide amino groups, respectively.

Figure 4.

Proposed mechanistic pathway for the synthesis of aminopyazolone derivatives 2-6.

Similarly, exchanging the nitrile functionality in compound 1 with the acetyl group in compound 7 to undergo the same combination of condensation reaction produced the targeted methyl pyrazolones 8–12 (Figure 5). The mechanistic discipline for this condensation was proposed to occur via nucleophilic addition of the first hydrazine amino group to the electrophilic ester carbonyl functionality pursued by proton transfer and lack of one mole of ethanol to yield the nonisolable intermediate. A synchronous nucleophilic addition of the second NH₂ function to aceto carbonyl functionality and intramolecular cyclization with removal of H₂O moiety gave rise to the formation of methylpyrazolones 8–12 (Figure 5). The affirmation for the formation of supposed structures was considered via ¹H NMR spectra that lacked triplet and quartet signals of ethyl carboxylate moiety and revealed the existence of D₂O exchangeable singlet at 11.59 ppm due to pyrazole NH group in compound 8. Furthermore, the existence of D₂O exchangeable singlets at 7.14 and 9.44 ppm in 10 and 11, corresponding to carboxamide and carbothioamide amino groups, respectively, supported the suggested structures. Finally, ¹³C NMR spectra of entities 8–12 showed signals for their representative carbons at their expected chemical shift.

Figure 5.

Synthesis of methylpyrazolone derivatives 8-12 and their suggested mechanism.

3.1.1. A comparative study between the yields of conventional, grinding & microwave-aided techniques

To obtain the target products in both series, the synthesis was carried out using conventional, grinding, and microwave-assisted techniques [30,56,57]. The achievement of the reaction was guided by TLC. Despite the molar ratios of the reactants being identical in each approach, the yields and the times required for each method to complete the reaction varied significantly. The variations in yields and reaction times for target compounds using conventional, microwave-assisted, and grinding methods are displayed in Table 1. To determine whether the method is more suited for this reaction, yield economy (YE) is used.

Table 1.

A comparison between conventional, grinding and microwave-assisted techniques.

The determination of YE is applied according to the following equation:

$$\mathrm{YE}=\frac{\mathrm{yield}\%}{\mathrm{Reaction time }“min”}$$

Also, reaction mass efficiency (RME) is detected in the three strategies.

$$\text{RME}=\frac{\mathrm{Wt of isolateted product}}{\mathrm{Wt of reactants}}$$

Another parameter for comparing the three approaches is optimum efficiency (OE), which can be calculated according to the following equation:

$$\mathrm{OE}=\frac{\mathrm{RME}}{\mathrm{AE}}\times 100$$

AE is assigned for the atomic economy. As a result, YE was used as a benchmark to compare the effectiveness of various techniques for creating an identical compound. It is worth mentioning that AE is the same for the three different techniques in which targeted compounds are prepared.

3.2. Biological activity

3.2.1. In vitro cytotoxicity investigation

The synthesized analogs were subjected to screening against three distinct tumor cells, including liver (HepG2), colon (HCT116) and breast (MCF-7) carcinomas employing the MTT colorimetric assay. Cytotoxicity of all synthetic compounds was assessed at seven doses and compared with the reference drug doxorubicin. Table 2 displays the cytotoxicity in the form of IC₅₀ values of the prepared compounds for the tested tumor cell lines. The cytotoxicity findings revealed varying degrees of activity ranging from potent to weak. From these analogs, 2, 4 and 6 exhibited promising antiproliferative effects against all tested cancerous cells. Compound 2 showed the strongest inhibitory action toward the HCT116 cell line with an IC₅₀ of about one-half of Doxorubicin. It was then followed by MCF-7 and HepG2 cell lines, whose IC_50s were nearly a third that of Doxorubicin. Additionally, compound 4 displayed the greatest sensitivity to HCT116 followed by HepG2, and the lowest sensitivity to the MCF-7 cell line. Further, aminopyrazolone 6 exerted significant cytotoxicity toward both MCF-7 and HepG2 cells, whose potencies were halved in comparison to Doxorubicin, followed by HCT116 cells. The remaining compounds exhibited cytotoxic activity ranging from moderate to weak.

Table 2.

IC₅₀ values of new aminoazopyrazolones and those of doxorubicin against cancer and normal cells associated with selectivity indices and CDK9 inhibition of most active analogs.

Comp. No.	aIC50 (μM) ± SD						Selectivity index (SI)b			Enzyme assay
	R	R1	HepG2	HCT116	MCF-7	WI38	HepG2	HCT116	MCF-7	CDK9
1			67.43 ± 3.5c	74.92 ± 3.8c	56.19 ± 3.1c	ND	ND	ND	ND	ND
2	NH2	H	13.03 ± 1.0 c	8.94 ± 0.7 c	11.20 ± 0.9 c	62.82 ± 3.6c	4.82	7.03	5.61	2.108 ± 0.027d
3	NH2	C6H5	>100c	>100c	84.83 ± 4.3c	ND	ND	ND	ND	ND
4	NH2	CONH2	21.06 ± 1.6 c	17.33 ± 1.4 c	26.54 ± 2.0 c	37.70 ± 2.1c	1.80	2.18	1.42	7.149 ± 0.008d
5	NH2	CSNH2	33.08 ± 2.1c	24.72 ± 1.9c	29.91 ± 2.1c	ND	ND	ND	ND	ND
6	NH2	COC6H5	9.31 ± 0.7 c	15.15 ± 1.2 c	7.58 ± 0.5 c	43.09 ± 2.5c	4.63	2.84	5.68	0.496 ± 0.056
7			76.66 ± 3.8c	>100c	68.80 ± 3.6c	ND	ND	ND	ND	ND
8	CH3	H	48.23 ± 2.7c	51.48 ± 2.9c	36.53 ± 2.2c	ND	ND	ND	ND	ND
9	CH3	C6H5	88.38 ± 4.2c	92.34 ± 4.7c	73.81 ± 3.9c	ND	ND	ND	ND	ND
10	CH3	CONH2	39.25 ± 2.2c	35.33 ± 2.1c	30.97 ± 2.3c	ND	ND	ND	ND	ND
11	CH3	CSNH2	54.55 ± 3.1c	63.91 ± 3.5c	42.61 ± 2.4c	ND	ND	ND	ND	ND
12	CH3	COC6H5	61.44 ± 3.3c	58.69 ± 3.2c	47.40 ± 2.6c	ND	ND	ND	ND	ND
Doxorubicin	–	–	4.50 ± 0.2	5.23 ± 0.3	4.17 ± 0.2	6.72 ± 0.5	1.49	1.28	1.61	ND
Dinaciclib			ND	ND	ND	ND	ND	ND	ND	0.053 ± 0.003

^aResults are the average ± standard deviation of three repeated experimental assays.

^bSI = IC₅₀ (normal cells)/IC₅₀ (tumor cells); SI: ≥5 (very selective), >2 (moderately selective) and <2 (not selective).

The statistical significance was assessed by one-way ANOVA followed by Turkey post hoc test.

^csignificantly different from doxorubicin at p < 0.05.

^dsignificantly different from dinaciclib at p < 0.05.

ANOVA: Analysis of variance; ND: Not detected.

3.2.2. Selectivity index

In vitro cytotoxicity of the most potent designated compounds 2, 4 and 6 toward the nontumorigenic WI-38 cells was further evaluated in order to examine the safety of these molecules. As stated by Badisa and colleagues [58], the selectivity degree of compounds was assessed by calculating their selectivity index (SI) value. Compounds with SI value of 5 or greater indicated high selectivity, while values exceeding 2 suggested moderate selectivity. Conversely, SI values below 2 were indicative of nonselectivity. Table 2 presents the SI of active compounds 2, 4 and 6 toward HepG2, HCT116 and MCF-7 cells in comparison to the reference drug doxorubicin. According to the results, compounds 2 and 6 exhibited approximately three-times the selectivity of doxorubicin against HepG2 and MCF-7 tumor cells. Further, entity 2 demonstrated about fivefold selectivity against colon HCT116 cells when compared with doxorubicin. In contrast, compound 6 displayed a moderate degree of selectivity against the colon HCT116 cell line, which was about twofold that of doxorubicin. However, compound 4 had the lowest selectivity, which was comparable to doxorubicin.

3.2.3. CDK9 inhibition

CDK9 is crucial for regulating basal gene transcription and maintaining transcriptional homeostasis in cells. The pertinence of CDK9 in cancer is highlighted by its fundamental role in transcriptional programming, as manifested by the dysregulation of these programs. Such a perspective renders CDK9 a promising target for cancer treatment strategies [59]. Increased expression of CDK-9 is accompanied by the development of various tumors, such as breast and hepatocellular carcinomas [30]. Therefore, an evaluation was conducted to investigate the inhibitory activity against CDK9 of the most potent cytotoxic analogs 2, 4 and 6 utilizing Dinaciclib as the standard agent. Analog 6, which had a benzoyl moiety at N-1 of aminopyrazolone scaffold, displayed remarkable CDK9 suppression with an IC₅₀ equal to 0.496 μM, as illustrated in Table 2. Meanwhile, entity 2, having nonsubstituted aminopyrazolone, presented considerable inhibition, as showed by its IC₅₀ value of 2.108 μM. Furthermore, the amide-containing compound 4 demonstrated mild inhibitory activity, exerting an IC₅₀ value of 7.149 μM. The present findings suggest that the CDK9 inhibitory effect of the designated aminopyrazolones may be influenced by the lipophilic and steric effects of the substituent on the endocyclic NH group. This observation merits further investigation and emphasizes the potential significance of such factors in a molecular design targeting CDK9 inhibition.

3.2.4. Structure activity relationship

The newly designated compounds are structurally classified as two series, namely aminopyrazolones (2–6) and methylpyrazolones (8–12). It was observed that the strongest cytotoxic agents were from compounds of the first series. Compounds 2, 4 and 6 with (R¹ = H, CONH₂ and COC₆H₅) showed significant cytotoxicity in comparison with Doxorubicin. The cyclization of compound 1 to aminopyrazolone 2, where R¹ denotes hydrogen, resulted in a high elevation in cytotoxic potency against three types of tumor cell lines with IC₅₀ values of 13.03, 8.94 and 11.20 μM toward HepG2, HCT116 and MCF-7 cells, respectively, comparing to Doxorubicin (IC₅₀ = 4.50, 5.23 and 4.17 μM). The cytotoxicity was completely abolished when a phenyl ring was added to the NH of aminopyrazolone, as in compound 3. Whereas the insertion of an amide moiety, compound 4, markedly boosted its inhibition activity in comparison to its precursor, especially against colon HCT116 cells. Further, the substitution of aminopyrazolone-NH by thioamide fragment, as in compound 5, displayed moderate cytotoxic activity. Whereas the incorporation of benzoyl moiety, the aminopyrazolone 6, led to robust antiproliferative activity as indicated by the IC₅₀ values of 9.31, 15.15 and 7.58 μM toward the tested tumor cells, as depicted in Supplementary Figure S1 in Supplementary files. On the other hand, analyzing the cytotoxicity outcomes of the second series revealed that the existence of a methyl functionality (having a positive inductive effect) rather than an amino functionality (with a positive mesomeric effect) at the C-3 position of the pyrazolone core had a negative impact, as the cytotoxic activity was remarkably decreased. In this series, compound 10 (R¹ = CONH₂) had the highest cytotoxic activity, with IC₅₀ values of 39.25, 35.33 and 30.97 μM against HepG2, HCT116 and MCF-7 cells, respectively. However, the remaining derivatives in the same series 7–9, 11 and 12 demonstrated varying degrees of cytotoxicity, ranging from mild to poor activity. Collectively, the involvement of NH₂ moiety at C-3 of the pyrazolone scaffold is essential for the cytotoxic activity, where the inclusion of hydrogen or carbonyl functionalities at N-1 enhanced the cytotoxic effect.

3.2.5. Cell cycle analysis

The MCF-7 cell cycle was analyzed to explore the mechanisms of action of the most potent cytotoxic agent with promising CDK9 inhibitory activity, compound 6. The introduction of compound 6 to MCF-7 cells resulted in a cessation of cell propagation at the G2/M phase, as indicated by a significant increase in cell count at this stage (22.06%) when compared with the control (9.85%). Additionally, a little reduction in the cellular count at S phase was detected, falling from 33.76 to 32.76%. Also, a marked decline in cellular percentage at G0/G1 phase was demonstrated, dropping from 56.39 to 45.18% compared with the control (Supplementary Figure S2 in Supplementary files). Consequently, aminopyrazolone 6 was shown to significantly alter the cell cycle profile and arrest the cell cycle at G2/M phase.

3.2.6. Cellular apoptosis screening

Compound 6 significantly induced cell apoptosis in MCF-7 cells when it was screened using annexin-V/PI protocol. In comparison to untreated cells, compound 6 highly increased the percentage of cells in the late apoptotic stage by 139.53-fold. Additionally, it showed a significant rise in the proportion of cells at the early and total apoptosis by 33.27 and 24-fold, respectively. Furthermore, aminopyrazolone 6 exhibited a 3.11-fold increase in the proportion of necrotic cells compared with nontreated cells (Supplementary Figure S3 in Supplementary files). Overall, the results of our study indicate that compound 6 may exert cytotoxic effects on cancer cells by arresting the cell cycle, specifically at the G2/M stage, as well as promoting apoptotic processes.

3.3. Molecular docking

A crystal structure of CDK9 complexed with the 3,5-diaminopyrazole-derived ligand has been downloaded from the official website of Protein Data Bank (PDB ID: 3TNH; Resolution: 3.20 Å; https://www.rcsb.org/structure/3TNH) [28,60]. The internal co-crystallized ligand (CAN-508) was re-docked, and twenty poses were requested. Also, similar runs were conducted on the reported potent CDK9 inhibitors (dinaciclib, seliciclib and alvocidib) as well as the new compounds that showed a good modulation effect on CDK9 in the in vitro studies. Docking processes were validated in terms of the RMSD [4,61]. Then, the obtained poses were scored, and the acceptable poses were chosen for further analysis.

Binding interactions of CAN-508 with CDK9 exhibited -5.26 kcal/mol as the free energy of binding and four ultimate hydrogen bond (HB) connections with the receptor, as presented in Supplementary Figure S4 in Supplementary files. A bidirectional HB was formed between the carboxylic acid group of the amino acid residue Asp104 and the pyrazolone-NH and NH₂ functionalities of CAN-508. A third HB connection has been observed between the pyrazole-N2 and the primary amine group of Cys106. One more HB connection has also been viewed between the residue Lys48 and the phenolic group of CAN-508.

The interaction pattern of dinaciclib with targeted protein released 13.54 kcal/mol. The anionic oxide of the ligand acted as an acceptor to form the first HB interaction with Gly31. In the second HB, the CDK9 inhibitor ligand donates a hydrogen atom to form one more interaction with Thr29. In different ways, seliciclib and alvocidib interacted with the crystal structure of the CDK9 through two connections for each. With -11.41 kcal/mol as free energy, seliciclib made two hydrophobic interactions with Ile25 and Asp167 amino acid residues. Two HB connections have been observed between alvocidib and Asp104 and Cys106 amino acid residues. The RMSD values for dinaciclib, seliciclib, and alvocidib were 1.20, 1.07, and 1.69 Å, respectively.

The new aminopyrazolones 2 and 6 showed much better free energies of bindings compared with the internal co-crystallized CDK9 inhibitor (-8.98 and 11.41 kcal/mol, respectively, compared with -5.26 kcal/mol). Also, each one of the new ligands has occupied the same pocket of CAN-508 and showed two HB connections. The selected pose of 2 exhibited RMSD value (1.38) and two HB connections. The first one has been observed between one of the sulphaguanidine amino groups and the carbonyl oxygen of the residue Asp167 within 2.87 Å. In the second connection, the side chain NH₂ functionality attached to C-3 of the pyrazolone fragment interacted with crucial amino acid Cys106 within a distance of 3.30 Å. In the case of the selected docking pose of the best effective CDK9 inhibitor 6, the RMSD value was found to be 1.27. Once again, the side chain NH₂ functionality attached to C-3 of the pyrazolone fragment of 6 interacted with the amino acid Asp104 within a distance of 3.05 Å. Additionally, another HB connection has been detected within less than 3.00 Å between Asp109 and one amino group of the sulphaguanidine moiety. These connection patterns as well as the higher binding scores, especially for compound 6, may further rationalize the good results obtained in the in vitro tests [62,63]. An outline of binding energies, RMSD values, HB and connection distances of the new candidates 2 and 6 are given in Table 3 & Supplementary Figure S4 in Supplementary files.

Table 3.

Binding energies, root mean square deviation values, hydrogen bonding connections and connection distances of the new aminopyrazolones 2 and 6, in addition to reference CDK9 inhibitors with CDK9.

Compound number	Score (Kcal/mol)	RMSDa (Å)	Amino acid residue	Type	Distance (Å)
CAN-508	-5.26	1.07	Asp104	HB-donor	2.99
			Asp104	HB-donor	2.70
			Lys48	HB-acceptor	2.50
			Cys106	HB-acceptor	3.15
Dinaciclib	-13.54	1.20	Gly31	HB-acceptor	2.23
			Thr29	HB-donor	2.08
Seliciclib	-11.41	1.07	Ile25	Arene-H	4.36
			Asp167	Arene-H	4.29
Alvocidib	-13.35	1.69	Asp104	HB-donor	2.88
			Cys106	HB-acceptor	3.41
2	-8.98	1.38	Asp167	HB-donor	2.87
			Cys106	HB-donor	3.30
6	-11.41	1.27	Asp109	HB-donor	2.99
			Asp104	HB-donor	3.05

^aRMSD: Root mean square deviation.

3.4. In vivo biodistribution studies

Recently, we reported diaminopyrazoles XI as significant cytotoxic and CDK9 inhibitors [30]. Herein, we study the structural optimization of these compounds toward the identification of new CDK9 inhibitors with improved pharmacokinetic and biodistribution properties. The aminopyrazolone 6 was selected as the most potent CDK9 inhibitor for the comparative tumor uptake study between XI and 6 utilizing the radiolabeling technique.

3.4.1. Radiochemical efficiency (RE) of radioiodinated compounds 6 & XI

In order to study the variation of radiochemical efficiency (RE), five parameters were varied at different ranges including compound amount (50–250 μg), chloramine-T amount (50–250 μg), reaction time (10–60 min), reaction temperature (25–100°C) and pH (2–11) (Supplementary Figures S5 & S6 in Supplementary file). The highest RE of radioiodinated compound 6 was about 98.5 ± 0.8%. The maximum yield could be obtained by utilizing 150 μg of analog 6 and 150 μg of chloramine-T (CAT). The radiolabeling reaction was executed at 25°C for half an hour. On the other hand, the RE of XI was 96 ± 0.7% and the highest yield was obtained by utilizing 150 μg of XI and 200 μg of CAT at 40°C for 30 min. To assess data differences, a one-way Analysis of variance statistical test was employed, with a significance level of p < 0.05. Up to 24 h, the radiolabeled compounds demonstrated good in vitro stability.

3.4.2. Biodistribution study of ¹³¹I-compound 6 & ¹³¹I-compound XI in normal & tumor-induced mice

The biodistribution patterns of ¹³¹I-compound 6 & ¹³¹I-compound XI were studied and analyzed in both normal and tumor-induced mice at different time intervals (0.5, 1, 2 and 4 h) after injection. Results of biodistribution in various bodily tissues, organs, and fluids are shown in Supplementary Figure S7 in Supplementary file. It is to be noted that both compounds were excreted through the renal route. Furthermore, lower thyroid uptake values might be an indication of the stability of radiolabeled compounds in vivo [64]. A comparison of the tumor uptake of both radiolabeled compounds is presented in Supplementary Figure S8 in Supplementary file. It is clear that ¹³¹I-compound 6 displayed the greatest significant (p < 0.05) tumor uptake, with an injected dose (% ID/g) of 27.3 ± 0.03% after 2 h of intravenous injection, while ¹³¹I-compound XI presented 17.2 ± 0.02 after 2 h of intravenous injection. Additionally, 2 h after intravenous injection of ¹³¹I-compound 6, the tumor uptake/blood (T/B) ratio was also considerably greater (p < 0.05) than that of ¹³¹I-compound XI (3.5 ± 0.09 and 2.1 ± 0.07, respectively). This finding further supports the selectivity and potentiality of ¹³¹I-compound 6 for targeting the tumor.

3.5. In silico toxicity prediction

The in silico toxicity of the most potent pyrazolones, as antitumor agents, was carried out utilizing the pkCSM predictor [65] compared with two references, doxorubicin and dinaciclib. The outcomes showed that compounds 2 and 6 are predicted to be nongenotoxic as deduced from no AMES toxicity test. Furthermore, compounds 2 and 6 possess the same benefits as doxorubicin and dinaciclib in that they do not block hERG I, meaning they do not cause cardiotoxicity side effects. Although compound 2 is superior to doxorubicin and dinaciclib in its cardiac safety suggested by being hERG II noninhibitor, compound 6 has the same drawback as the reference antitumor drugs, which is a positive probability of cardiac arrhythmia as shown in hERG II inhibitory test. Interestingly, the oral acute toxicity of 2 and 6 are nearly identical or lower than that of the reference drugs. As well, compounds 2 and 6 presented much higher tolerability than doxorubicin due to their lower oral rat chronic toxicity. Moreover, the two tested compounds and two references shared the advantage of no skin sensitization while they have the identical disadvantage of the predicted hepatotoxicity. Additionally, compounds 2 and 6 displayed lower Minnow toxicity than doxorubicin and almost similar Pyriformis toxicity to doxorubicin and dinaciclib. Detailed results of the in silico toxicity predictions are illustrated in Supplementary Table S1 in the Supplementary file.

4. Conclusion

Deeming the exemption of nonfundamental HBD groups as a crucial strategy in medicinal chemistry, the structural optimization of the last reported series of sulfaguanidine-linked 3,5-diaminoazopyrazoles and azopyrazolidine-3,5-diones toward the exploration of novel anticancer and CDK9 inhibitors containing aminopyrazolone and methylpyrazolone scaffolds with improved pharmacokinetic properties was accomplished. The synthesis of the target compounds was adopted through three approaches (conventional, grinding and microwave-assisted processes). The yields that were produced and the times required for complete reactions in the three approaches varied significantly. Three compounds from aminopyrazolone series 2, 4 and 6 exhibited significant cytotoxic activity and selectivity toward all tested cancerous cells, presenting an IC₅₀ range of 7.58–26.54 μM and SI range of 1.42–7.03, respectively. They also displayed potent CDK9 inhibition with an IC₅₀ range equal to 0.496–7.149 μM. The superior cytotoxic and CDK9 inhibitor 6 underwent further estimation to determine its mode of action. Therefore, the aminopyrazolone 6 arrested the MCF-7 cell cycle progression at the G2/M phase via stimulating the apoptotic pathway. Studies on molecular modeling illustrated a distinctive interaction between CDK9's active pocket and compound 6. The optimized stable radiolabeled compound 6 succeeded in targeting the tumor, as evidenced by a higher ratio of tumor uptake/Blood (T/B) of 3.5 ± 0.09 compared with previously reported XI with a (T/B) ratio of 2.1 ± 0.07. Additionally, compound 6 succeeded in acting as a chemical carrier for radioactive iodine (¹³¹I), indicating considerable potential for serving as a significant radiopharmaceutical agent for the imaging and therapy of cancer.

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17568919.2024.2419363

Financial disclosure

This paper was not funded.

Competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Ethical conduct of research

Every procedure involving animals was conducted according to the guidelines approved by the Ethics Committee for Experimental Studies (Human& Animal subject) at the National Center for Research Radiation and Technology-Egyptian Atomic Energy Authority, Cairo, Egypt (9A/24).