A Comprehensive Overview of the Developments of Cdc25 Phosphatase Inhibitors

Cdc25 phosphatases have been considered promising targets for anticancer development due to the correlation of their overexpression with a wide variety of cancers. In the last two decades, the interest in this subject has considerably increased and many publications have been launched concerning this issue. An overview is constructed based on data analysis of the results of the previous publications covering the years from 1992 to 2021. Thus, the main objective of the current review is to report the chemical structures of Cdc25s inhibitors and answer the question, how to design an inhibitor with better efficacy and lower toxicity?


Introduction
The cell division cycle 25 (Cdc25) phosphatases are dual-specificity phosphatases (DSPs) that catalyze the dephosphorylation of the Cdk/Cyc protein complex, an important regulator of the human cell cycle [1]. In human cells, three Cdc25 phosphatases are characterized, Cdc25A, Cdc25B, and Cdc25C, which share the similarity of amino acids identity from 20 to 25% for N-terminal and 60% similarity for C-terminal, and are differentially expressed in the cell division cycle [2,3].
Cdc25A is involved in the control of the G1/S transition by dephosphorylating and thus activating Cdk2/cyclin E and cyclin A complexes, as well as controlling the progression into mitosis. Additionally, Cdc25A activates Cdk1/cyclin B provoking the transition G 2 /M [4], while Cdc25B and C mainly regulate the progression at the G2/M transition and mitosis [4,5]. Cdc25B accumulates from the late S and early G2 phases of the cell cycle and its activity peaks at the G2/M transition [4,5]. Cdc25B is proposed to play a "starter" role in triggering mitosis by dephosphorylation and thereby activating Cdk1/cyclin B at the centrosome level [4,5]. On the other hand, Cdc25C is mainly involved in controlling progression into mitosis and this activation is correlated to Cdc25C phosphorylation by Cdk1/cyclinB substrate [5]. The Cdc25 phosphatases also play a crucial role in the checkpoint response that prevents Cdk/cyclin activation following DNA damage [6,7].
Many studies have shown that Cdc25 is highly expressed in cancer. Therefore, it is considered an excellent target for cancer therapy [5,8,9]. Thus, inhibition of these phosphatases may represent a promising therapeutic approach in oncology [10][11][12].
This review is dedicated to searching for safe and efficient Cdc25s inhibitors derived from natural, synthetic, and computational sources. It is noteworthy that the thiolate of the active cysteine (Cys473 in Cdc25B) is found to have an extremely high rate of conversion to sulfenic acid. When it is tested with hydrogen peroxide, the result for Cdc25B is 15-fold and 400-fold faster than that for the protein tyrosine phosphatase PTP1B and the cellular reductant glutathione, respectively. This suggests that Cys426 has a preventing role in the formation of sulfenic acid (Cys-SO 2 ) in the Cdc25s by forming a rapid intramolecular disulfide linkage with catalytic cysteines (Cys473). This intramolecular disulfide is reversibly and rapidly reduced by cellular thioredoxin/thioredoxin reductase. Thus, the chemistry and kinetics of the activesite cysteines of the Cdc25s support a physiological role for reversible redox-mediated regulation of the Cdc25s as important regulators of the eukaryotic cell cycle [14].

Molecular Studies of Cdc25 Phosphatase
The crystal structure of the catalytic domain of Cdc25B displays a well-ordered active site containing a bounded sulfate. This active site loop creates a special environment for cysteine 473 [18,19]. Despite the lack of crystallographic structure in the protein-inhibitor complex and the nonexistence of a deep active site pocket, one of the largest cavities on the surface of Cdc25B adjacent 0.6 Å to the active site reveals good binding envelop for the small inhibitory molecules during docking processes ( Figure 2) [18]. The residues that surround the inhibitor binding pocket adjacent to the active site contain Arg482, Arg544, and Thr547 besides other residues [19].

Interaction between Cdc25 Phosphatase and Its Protein Substrate
Several investigations have tried to predict a docked orientation for Cdc25B with its Cdk2-pTpY-CycA protein substrate using different docking techniques [20]. The available crystal structure containing a bound sulfate (1QB0) at the active site is expected to be a good representative of the phosphate group of the phosphorylated protein substrate [21]. However, on the Cdk2-pTpY-CycA substrate, the exact orientation of the β-hairpin loop encompassing the adjacent Thr14 and Tyr15 sites of phosphorylation is unknown. The second point of contact between Cdc25B and its protein substrate is hot-spot residues (Arg488 and Tyr497). They are >20 Å from the active site and expected to make a contact with either Cdk2 or cyclin A at the binding interface [20].
Computational docking of Cdk2-pTpY-CycA to the enzyme followed by refinement using molecular dynamics was performed. In addition, validation of the docking using X-ray crystallography was applied to the crystal structure of the substrate-trapping mutant of Cdc25B [22,23]. The phosphate of pThr14 is cradled within the active site loop with numerous hydrogen bonds to the amides backbone including the side chain of Arg479 of the CX5R signature motif. This phosphate is additionally coordinated in the active site pocket by contributions from the amide backbone of Thr14 and the side chain of Arg36 from the substrate [20].
Further studies have been conducted to clarify the exact model for enzyme-substrate interaction. Some of these studies used small molecules such as p-nitrophenyl phosphate (pNPP) as substrate and applied the recently developed QM/MM Minimum Free Energy Path method. This was intended to gain an understanding of the dephosphorylation mechanism [1].

Cdc25 Inhibitors
A diverse number of structural inhibitors of the Cdc25 phosphatases have been identified and reported in the literature. Many of the Cdc25 inhibitors have been discovered via either the isolation of new scaffolds by High Throughput Screening (HTS) or optimization of pre-existing inhibitor scaffolds [24].

Cdc25 Inhibitors Based on the Quinoid Structure
Menadione (vitamin K 3 ) (1) is one of the first para quinoid compounds that proved its activity against Cdc25 A, B, and C (Ki: 38 ± 4, 95 ± 3, and 20 ± 4 µM, respectively). Suggestions to modify the structure by delivering polar derivatives of this nucleus did not lead to increased activity in contrast to what was expected [10,11].
The fluorinated analog 3 of Cpd-5 (2) had advantages over its parent nucleus since it was less toxic. The inductive effect of fluorine atom resulted in the liberation of lower ROS amount, thus lowering the toxicity. Meanwhile, it had higher potency with IC 50 of 0.8, 1, and 50 µM against Cdc25A, B, and C, respectively [27].
Another correspondence to vitamin K is Cpd-42 (4) and its isomer 5. Both had the same IC 50 toward Cdc25A (1.5 µM), which may explain the role of these compounds in the suppression of a Hep3B hepatoma cell line [28].
New biotin-containing Cpd-5, derivative 6, and its isomer 7, were found to be Cdc25 inhibitors by the arylation of the active cysteine of Cdc25A, B, and C. Cpd-5 derivative 6 and its isomer 7 exhibited a selective manner of inhibitions with IC 50 of 1.5, 30, and 100 µM for Cdc25A, VHR, and PTB1B, respectively. Carbonyl oxygens of the derivative appeared to interact with both Arg482 and Arg544 near the catalytic cysteine 473 [29].
On the other hand, the naphthalene-type analog of vitamin K 3 11 had Cdc25A phosphatase-inhibitory activity equal to IC 50 0.4 µM [31]. In addition, naphthoquinone derivatives, containing malonic 12 or carboxylic acids 13 groups were introduced to mimic the role of phosphate moieties of Cdk complexes. The malonic acid derivative 12 did not show the expected rise of the activity with IC 50 value 10.7 ± 0.5 µM on Cdc25B and displayed moderate cytotoxicities against the HeLa cell line [32], whereas compound 13 exhibited IC 50 of 4.55 ± 0.16 µM toward Cdc25B and weak activity against HeLa cell lines [32]. Bis(methoxycarbonylmethylthio)heteroquinone analog of vitamin K 3 14 showed reasonable activity against Cdc25B with IC 50 of 1.17 ± 0.04 µM [33].
The reversibility of the aforementioned compound was tested by the mass spectrometry (MALDI-TOFMS) technique and was proved to reversibly interact with Cdc25 A and C ( Figure 3, Table 1) [35].  A new family of potent inhibitors 16-28 of the dual-specificity protein phosphatase Cdc25 of quinone structure and analogs of vitamin K 3 was reported ( Figure 4, Table 2). 6-Chloro-7-(2-morpholin-4-ylethylamino) quinoline-5,8-dione (NSC 663284) (16) was described as a very potent irreversible inhibitor of Cdc25 A, B with IC 50 : 210 nM; additionally, 16 showed 20-and 450-fold selectivity for Cdc25B compared to VHR or PTP1B phosphatases, respectively [36]. The NSC 663284 (16) showed a direct binding to one of the two serine residues in the active site loop of the Cdc25A catalytic domain (Ser114, corresponding to Ser434 in full-length Cdc25A) [36]. Similar compounds JUN 1111 (17) also acted by induction of ROS, which led to irreversible oxidation of the catalytic cysteine thiol to sulfonic acid; therefore, cells were arrested in G1 and G2/M phases of the cell cycle. Enzyme suppression was found to be affected by medium pH, in the presence of catalase and reductants (dithiothreitol and glutathione) [37]. Quinone structures 18, 19, 20 were reported to have IC 50 toward Cdc25B of 0.5 ± 0.1, 1.1 ± 0.1, and 5.3 ± 0.6 µM, respectively. They were designed to have half-wave potentials less than 105 mV, to obtain a stable molecule in its reduced state. Theoretically, it would produce a controlled amount of ROS or H 2 O 2 . Unfortunately, it was found that their redox cycle and E1/2 did not match what was expected [38].
Caulibugulones A-F (21-26) are isoquinoline derivatives obtained from the marine bryozoan Caulibugula intermis exhibiting a IC 50 values range of 2.7-32.5 µM towards Cdc25B in very intensive selectivity compared with other phosphatases VHR and PTP1B [39]. Caulibugulones A-F (21-26), displayed distinct patterns of differential cytotoxicity against 60 antitumor cell lines. The results showed that 21-26 revealed in vitro cytotoxicity with IC 50 values in the range of 0.03-1.67 µg/mL against murine tumor cell line IC-2 WT [40,41]. By investigating the inhibitory mechanism of caulibugulone A (21), it was found that its inhibition effect was not attributed to the release of ROS, as caulibugulone A (21) releases a smaller amount of ROS. The inhibition appeared to take place by the stress-activated protein kinase p38 pathway. This protein controls mitosis under the condition of osmotic pressure by degradation of Cdc25A through its phosphorylation at Ser75. Caulibugulone A (21) activated p38 which leads to degradation of Cdc25A with little or no loss of either Cdc25B or C [41]. (27) showed IC 50 of 1.63 ± 0.21 µM on Cdc25B. In addition, 27 showed inhibition percent of 79 ± 0.9 and 77 ± 2.1% at 100 µM toward HeLa and MiaPaCa-2 cell lines, respectively, while at 10 µM it showed an inhibition of 13 ± 0.9 and 24 ± 15.4% against the same cell lines, respectively [33].
One of the earlier Cdc25 inhibitors of quinoid scaffold, which naturally originated from Streptomyces sp., is 40 which showed inhibition activity against Cdc25A with IC 50 of 15.5% at 10 µM, recording the most potent activities among the isolated compounds [50] ( Figure 6).
The most powerful quinone that has ever been discovered is adociaquinones B (41) which was separated from Xestospongia sp. with IC 50 of 0.08 ± 0.01, 0.07 ± 0.01 µM against Cdc25A and the B phosphatase catalytic domain [51] (Figure 6). Simplified adociaquinone B analogs 42 and 43 were synthesized and showed IC 50 of 0.94 and 0.88 µM against Cdc25B, respectively. When they were tested against human ovarian cell line A2780 they showed IC 50 of 4.3 ± 2.3, 2.1 ± 0.02 µM, respectively ( Figure 6). They have an advantage over adociaquinone B in their smaller size, which makes them more accessible to the binding site and also gives versatility for derivatization [52].
Compound 49 was intended to be applied as the precursor of 50, but surprisingly it was found to be much more active than the compound of focus ( Figure 6). Interestingly, 49 achieved inhibition against Cdc25A (IC 50 = 2.1 µM, 25-fold higher potency than B and C isoforms). It also induced apoptosis in human hepatoma cell line Hep3B with IC 50 of 2.5 µM representing 2.5-fold higher potency than Cpd-5, and 3.2-fold higher potency than Vitamin K 3 without harming the normal cells as it did not generate ROS [57,58]. Non-quinone Cpd 5 analog 50 was synthesized in order to obtain a non-quinoid structure to produce less or no ROS. It exhibited an IC 50 value of 7.5 µM against Cdc25A.
Quniolinone 52 may be considered as an isosteric analog to quinolinequinone with partial quinoid character ( Figure 6). Quniolinone 52 Was produced after several synthetic steps and inhibited Cdc25 phosphatase at 5 µM. Additionally, it was bioassayed with different cell lines but did not appear to have potential activity [60].

Cdc25 Inhibitors Based on Steroid and Dysidolide-like Compounds
Dysidiolide 53 was the first naturally discovered Cdc25A phosphatase inhibitor (IC 50 9.4 µM) with antitumor activity [61]. The γ-hydroxybutenolide group may surrogate the phosphate moiety of the substrate. The hydrophobic side chain is suggested to fill the hydrophobic groove adjacent to the active cysteine [62]. On contrary, Blanchard et al., suggested that the activity of dysidiolide was related to the other component in the crude extract not to the dysidiolide itself [63,64]. The IC50's of their synthetic dysidiolide were high (35 and 87 µM towards Cdc25A and Cdc25B, respectively); additionally, it showed an inhibitor activity of 5.4 and 7.1 µM against SBC-5 and HL60 cell lines [64].  [64].
Coscinosulfate 65 was extracted from marine sponge Coscinoderma matthewsi and showed the best activity among its synthetic analogs on Cdc25A phosphatase with IC 50 of 3 µM [75].
2-Methoxyestradiol (2-ME) 66 suppressed the growth of hepatoma cells by IC 50 of 0.5-3 µM without affecting the normal hepatic cell of the rat up to a concentration of 20 µM. The inhibition mechanism was suggested to happen by competitive inhibition of Cdc25s on the catalytic cysteine using biotin-labeled Cpd 5 (Cpd 5-Bt) as a ligand. The measured IC 50 0f 65 was 1, 1, and 10 µM for Cdc25A, B, and C [76].
The Chemical structures of compounds 53-66 and their biological activity are illustrated in Figure 7 and Table 4.

Cdc25 Inhibitors Based on Glycosidic Structures
Some glycosidic derivatives were proved to be potential inhibitors ( Figure 8)

Miscellaneous Cdc25 Inhibitors
The synthetic approach of RK-682, isolated from Streptomyces sp., led to compound 70 which is an R stereoisomer that inhibited Cdc25A and B with equal potency of 34 µM. It was more potent against VHR with IC 50 of 3.4 µM and not active against protein serine/threonine phosphatase l (PP1) [80]. Another example is compound 71 which showed activity with IC 50 of 0.38 ± 0.02 µM against Cdc25B [81].
To replace the resonating negative core of RK-682 with a neutral one as a suggestion to increase cell permeability and selectivity, the enamine 72 (RE44) was developed. It revealed suppression of Cdc25A (IC 50 : 13.5 ± 3.4 µM) and Cdc25B (IC 50 :4.26 ± 0.17 µM). No production of ROS was recorded [82].
In a patent released in 2002 by Jill et al., an intensive effort was exerted to identify peptidic inhibitors. They combined X-ray and computational methods to identify the 3-D image of amino acids in the natural substrate of the Cdc25 enzyme. Combinatorial chemistry was utilized to synthesize a wide variety of peptidic compounds comprising the crucial amino acids moieties defined in the substrate as reliable for binding with the Cdc25 enzyme. Compound 76 Is a pentapeptide invented by the above-mentioned technique with IC 50 of 1.1 µM against Cdc25A [87].
Compound 77 is a thiazolidinone derivative. Its activity as a selective Cdc25A inhibitor was reported in a comparative analysis with MKP-1 by stopping the cell cycle in Go/Gi or S phases. On the other hand, 77 provided cytotoxic activity against different human cancer cell lines including MDA-MB-435, PC-3, A549, and Bx-PC3 [88].
Poly prenyl furanes 81 and hydroquinones 82 were isolated from three sponge species and demonstrated potential activity towards Cdc25A phosphatase with IC 50 of 2.5 and 0.4 µM [92].
Compounds 87 and 88 were structurally inspired by suramin. Compound 87 showed high potency with IC 50 equal to 0.38 ± 0.04 on Cdc25A but with no selectivity, whereas compound 88 showed IC 50 of 3.2 ± 0.3 on the same target but with great selectivity versus other phosphatases [94].
Fascaplysin 89 is an alkaloid isolated from Thorectandra sp. sponge and revealed an IC 50 value of 1.0 µg/mL against Cdc25B. Sesterterpenoids 90 was obtained from the previously mentioned organism and demonstrated IC 50 (µg/mL) of 1.6 on Cdc25B [95].
Diterpenoid 91 was obtained from a sea anemone and revealed an inhibitory activity against Cdc25B with IC 50 of 1.6 µg/mL [96].
Some dimers of cinnamaldehyde were synthesized by Shin  Cheon et al., invented a large number of thiazolidine derivatives, and they evaluated the efficiency of one of them (93). Compound 93 was examined against Cdc25B and some cancerous cell lines, namely A-549, HT29, and MCF-7. The IC 50 of this compound, when incubated with the protein, was 2.26 µM and it appeared to be more active against the previously mentioned cell lines in comparison with the reference at 10 µM with IC 50 of 67.5, 59.7, and 67.3%, respectively [98].
Ryu and his coworkers identified 99 and 100 as novel Cdc25 phosphatase inhibitors with micromolar activity utilizing a structure-based de novo design method. The IC 50 s of both structures toward Cdc25A and B were 5.1, 1.2, 4.7, 2.3 µM, respectively [103].
A coumarine-based structure was first introduced as a potential Cdc25 inhibitor scaffold in 2010; 103 was the most active hit among other derivatives which were under investigation. The activity of 103 exhibited IC 50 of 27 and 29 µM towards A and B isoforms, and it inhibited Cdc25A, B, and C by percentages of 94.2 ± 7.3, 35 ± 5.5, and 79.3 ± 2.8, respectively at 100 µM [106].
Using HTS techniques, Choi and his coworkers discovered a non-peptide, small inhibitor of PTP-1B that serves as an oral glucose-lowering agent. Among them, the KR61170 (B) 104 has shown PTP-1B inhibitory activity of IC 50 equal to 0.29 ± 0.1, as well as Cdc25A, B inhibitory properties with IC 50 of 1.5 ± 0.3 and 0.17 ± 0.1 µM, respectively [107].
In 2009, The University of Pittsburgh Molecular Library Screening Center (Pittsburgh, PA, USA) conducted a screen with the NIH compound library for inhibitors of Cdc25B. Six of the nonoxidative hits were selective for Cdc25B inhibition versus MKP-1 and MKP-3. Only the two bisfuran-containing hits, (105) and (106), significantly inhibited the Cdc25B with IC 50 's of 11.6 and 15.5 µM, respectively. In addition, they significantly inhibited the growth of the MBA-MD-435 breast and PC-3 prostate cancer cell lines with IC 50 's of 96.2 ± 68, 15.3 ± 1.36, 50.4 ± 8.6, and 47.5 ± 2.75 µM, respectively. They suppressed enzymes in a mechanism that appeared to be not related to H 2 O 2 or ROS production, which makes it safer and interesting for further investigation [108].
The well-known antitumor activity of coumarin and its diallyl polysulfides was the spark of the synthesis of new dicoumarine products linked together with di, tri, and tetra sulfide linkage. This group of derivatives exhibited activity against HCT116 cell lines in a time and dose-dependent manner. The reduction in cell viability was 30%, 22%, and 18% for 108, 109, and 110 when treated with 25 µM and to 50% in the concentration of 50 µM. ROS release was the proposed mechanism of cellular inhibition as it happened at the G2/M phase which is mainly regulated by Cdc25C phosphatase [110]. Compounds 108, 109, and 110 decreased the level of Cdc25A and C, and this activity was proportional to the increasing number of the sulfur atoms but less than the activity of BN82002 (79) [110].
Early published work by Lauria et al.,identified (115,J3955) as Cdc25 modulators by the correlation between the chemosensitivity of J3955 and the protein expression pattern of the target enzyme. J3955 (115) showed concentration-dependent antiproliferative activity against HepG2 cells with GI 50 of 1.50 ± 0.37 µM. Western blotting analysis showed an increment of phosphorylated Cdk1 levels in cells exposed to J3955, indicating its specific influence in cellular pathways involving Cdc25 A, B, and C proteins with IC 50 's of 1.12 ± 0.09, 2.19 ± 0.07, and 2.22 ± 0.07 µM, respectively [114]. The mentioned miscellaneous Cdc25 inhibitors 70-115 are represented in Figure 9.

Computational Methods to Discover and Optimize Cdc25 Inhibitors
Molecular modeling and other computational techniques have been applied for screening of large libraries of compounds or for much greater comprehension of the mode of interaction of already proven active compounds.
One of the earlier molecular modeling computational analyses was applied for vitamin K 3 derivative (Cpd5,2) with Cdc25A phosphatase. Cpd 5 (2) was found to be closer to the enzyme active site than VK 3 . It was bound with more favorable energy forming an adduct with the catalytic cysteine [25].
In 2006, Lavecchia et al., investigated the structural models for the interaction of various Cdc25B inhibitors namely 16 and 51 with the enzyme via AUTODOCK and GOLD computational docking programs [115]. In GOLD model, compound 51 was found to have dihydroxyquinone ring fitting in catalytic sites and 7-(2-methyl-benzyl)indolyl was placed between Arg482 and Arg544. The 4-carbonyl oxygen accepted the H-bonds from Arg479, and the 5-OH was involved in H-bonding with Phe475, Ser477, and Glu478. For compound 16 the more likely pose (AUTODOCK model) involved electrostatic interaction between 5,8-oxygen of the quinone and Arg544, Arg482, and Tyr428. The nitrogen atom of pyridine made a hydrogen bond with Arg482. The oxygen of the side-chain shared in this hydrogen bonds network with Phe475 and Arg479 ( Figure 10) [115]. In 2008, Park and his coworkers studied the mode of the interaction of NSC 95397 (8) by AUTODOCK and nanosecond molecular dynamics simulations. The molecule was fitted into the active site of Cdc25B forming hydrogen bonds with Glu478, Glu474, Arg544, and Tyr428. It was also found that the hydrophobic interaction plays a critical role in the strong stabilization of NSC 95397 (8) [116].
Another structure-Based virtual screening trial based on molecular modeling led to the discovery of some structures. Among them, 116 was the most potent with IC 50 of 0.82 ± 0.41 µM (Cdc25A) and 1.98 ± 0.44 µM (Cdc25B). The binding mode of 118 was determined using the available crystal structure of Cdc25B and a homology modeling of the Cdc25A structure. The orientation of the molecule in both enzymes was different due to the smaller active site of Cdc25A. Dihydroxyphenylcarbonyl moiety formed hydrogen bonds with the backbone of the enzyme while triazole moiety mimics the phosphate group of the substrate. The toluene group was stabilized by hydrophobic interaction with Trp507 and Arg501 in the Cdc25A which may explain the increased activity against it ( Figure 11) [117]. Another trial of structure-based virtual ligand screening computations was done with FRED, Surflex, and LigandFit, towards a compound collection of over 310,000 druglike molecules and the crystal structure of Cdc25B. Ninety-nine compounds were identified as promising Cdc25 inhibitors that also exhibited antiproliferative properties. In the same study, the biological analysis of 117 and 118 ( Figure 12) proved them as the most potent among the tested molecules (IC 50 of 13.0 ± 0.5 and 19 ± 1.3 µM) toward Cdc25B and (IC 50 of 15.8 ± 1.8 and 3.6 ± 1.2 µM) toward HeLa cells, respectively. The results previously obtained according to the bioassay did not match the computational analysis [118]. Using de novo drug design probed by LigBuilder program, structure 119 was identified ( Figure 12). It was tested on Cdc25A and B to give IC 50 equal to 2.3 and 1.7 µM, respectively. The mode of interaction with the enzyme was examined by AUTODOCK. It was found to be fitted in a slightly different way between Cdc25A and B. The terminal phenyl ring was inserted deeper in the larger active site of the B isoform and established hydrophobic interaction with Tyr428, Arg479, and Met531 which may describe the higher activity against this isoform. Cyano and furan moieties interact with both enzymes in a similar pattern of hydrogen bonds while the thiol group appeared to surrogate the phosphate of the substrate in both active sites [119].
A molecular field point strategy was recently employed by a Field Templater software package to scan the pharmacophores which are essential for the activity of reversible Cdc25s inhibition. Three derivatives of scaffolds thiazolopyrimidine, pyrazole, and naphthofurandione, which are known as reversible inhibitors, were used as templates to build up a pharmacophore model. They were used for scanning a wide library of compounds (~3.5 million) and introducing promising candidates which would be subjected to biological studies. Compound 120 (Figure 12) was an example of these products offered by this type of drug discovery. It exhibited IC 50 's of 2.7 ± 1.7, 1.7 ± 2.0, and 9.7 against A, B, and C isoforms, respectively, and 26.0 ± 1.0 on VHR [120].
In 2019, Ferrari and his co-workers conducted a pharmacophore-guided drug discovery program for the identification of scaffolds of the naphthoquinone group displaying inhibition of Cdc25. They found that 2-(2 ,4 -dihydroxyphenyl)-8-hydroxy-1,4-naphthoquinone (121, UPD-140) and 5-hydroxy-2-(2,4-dihydroxyphenyl) naphthalene-1,4-dione (122, UPD-176) were the most potent compounds that induced inhibition of CDK1 activity and blocked the mitotic transition followed by cell death (Figure 13). In mouse Apc/K-Ras mutant duodenal organoids, low doses of Cdc25 inhibitors caused the arrest of proliferation and expression of differentiation markers, whereas high doses induced cell death. In zebrafish embryos, used as in vivo xenograft models, the Cdc25 inhibitors led to tumor regression and reduction in metastases [121].

Conclusions
This review aims to summarize the present state-of-the-art regarding design of Cdc25 phosphatase inhibitors. The discovery of new Cdc25 inhibitors is extremely significant for cancer therapy due to the critical roles that these phosphatases play in regulating cell-cycle progression. From the studies mentioned in this review article, no certain scaffold can be identified as an exclusive Cdc25 phosphatase inhibitor. Many potent Cdc25 inhibitors were synthesized or isolated from natural extracts. Meanwhile, quinoids are considered the most potent inhibitors despite their toxicity. Quinoids act mainly as Michael acceptors for the negative charge of Cys-S − in the catalytic domain. It seems that the small size of quinoids allows them to be suited to the adjacent swimming pool which gives them a good chance to play their roles correctly. One of the promising perspectives for finding an efficient inhibitor may depend on the optimization of the already discovered quinoids to eliminate their toxicity without diminishing their activity. Molecular modeling and other computational techniques have been applied for screening large libraries of compounds and to shed the light on the mode of interaction of already proven active compounds.