Modular Synthesis and Antiproliferative Activity of New Dihydro-1H-pyrazolo[1,3-b]pyridine Embelin Derivatives

A set of new dihydro-1H-pyrazolo[1,3-b]pyridine and pyrazolo[1,3-b]pyridine embelin derivatives was synthesized through a multicomponent reaction from natural embelin, 3-substituted-5-aminopyrazoles and aldehydes. The synthesized compounds were evaluated against three hematologic tumor cell lines, HEL (acute erythroid leukemia), K-562 (chronic myeloid leukemia) and HL-60 (acute myeloid leukemia), and five breast cancer cell lines (SKBR3, MCF-7, MDA-MB-231, BT-549, HS-578T). The primate non-malignant kidney Vero cell line was used as the control of cytotoxicity. From the obtained results, some structure–activity relationships were outlined. Furthermore, in silico prediction of physicochemical properties and ADME parameters were determined for the derivatives with the best antiproliferative values.


Introduction
Heterocyclic compounds are of great importance in medicinal chemistry [1]. Molecules with these structures are present in many essential compounds for life as nucleic acids, amino acids, chlorophyll or vitamins, among others [2]. Titarenko et al. demonstrated during the development of the BioCore strategy [3] that more than 67% of the molecules included in the "Comprehensive Medicinal Chemistry" database contain heterocyclic rings. The non-aromatic heterocycles are twice as abundant as the heteroaromatics [4] and the combination of both is frequently present in alkaloids and drugs [5]. Among all heterocycles, nitrogenated heterocycles are particularly relevant in medicinal chemistry. Furthermore, an analysis of the approved drugs by the FDA database conducted by Njardarson et al. [6], revealed that 59% of them contained nitrogenated heterocycles. Pyrazoles are very attractive nitrogenated scaffolds since molecules having pyrazoles fused to other heterocycles such as dihydro-1H-pyrazolo [1,3-b]pyridines and pyrazolo [3,4b]pyridines have raised great interest due to the biological activities that they exhibit. Figure 1 shows some examples of antitumoral compounds that exhibit this type of structure such as spirooxindoles (A) with cytotoxic activity against triple-negative breast cancer MDA-MB-231 cell line [7], compound (B) with potent and selective inhibitory activity against FGFR kinase [8] and compound (C) as potent tubulin polymerization inhibition [9].
Although the use of MCR allows us a quick and easy access to molecules with complex structural motifs, one of the problems associated with the use of this kind of methodology is related to the selectivity in the formation of the reaction products. Consequently, when a synthetic strategy involving a domino mechanism is designed, it is of significant importance to evaluate the different functionalities of the reagents used, the possible reaction products, as well as the different reaction parameters in order to control the selectivity of the transformation. An example of the former is the work published by Kappe et al. where the multicomponent condensation reaction of 3-aminopyrazoles, with 1,3-diketones and aromatic aldehydes, allows the obtaining of different products under different reaction conditions, due to the presence of three non-equivalent nucleophilic centers in the aminopyrazole component [14].
Next, we proceeded to optimize the reaction conditions, focusing on achieving the highest yield to use small amounts of natural embelin due to the limited amount of it available in our laboratory. When the reaction was carried out under conventional heating using an equimolar ratio of the reagents (Table 1, entry 2), the product 4a was ob- Figure 1. Antitumoral compounds with a dihydro-1H-pyrazolo [1,3-b]pyridine or a pyrazolo [1,3-b]pyridine moiety.
Although the use of MCR allows us a quick and easy access to molecules with complex structural motifs, one of the problems associated with the use of this kind of methodology is related to the selectivity in the formation of the reaction products. Consequently, when a synthetic strategy involving a domino mechanism is designed, it is of significant importance to evaluate the different functionalities of the reagents used, the possible reaction products, as well as the different reaction parameters in order to control the selectivity of the transformation. An example of the former is the work published by Kappe et al. where the multicomponent condensation reaction of 3-aminopyrazoles, with 1,3-diketones and aromatic aldehydes, allows the obtaining of different products under different reaction conditions, due to the presence of three non-equivalent nucleophilic centers in the aminopyrazole component [14].
Next, we proceeded to optimize the reaction conditions, focusing on achieving the highest yield to use small amounts of natural embelin due to the limited amount of it available in our laboratory. When the reaction was carried out under conventional heating using an equimolar ratio of the reagents (Table 1, entry 2), the product 4a was obtained in 75% yield. Since heating was necessary in order to obtain a significant yield, we decided to test the use of MW irradiation to improve yields by shortening the reaction time and minimizing the side products. Additionally, we tried to use an alternative solvent, and modify the ratio of the reagents. In this sense, we selected DCE since in a previous work with embelin the use of DCE under MW irradiation produced very good results [19]. Considering this, it was found that the combination of DCE, and an excess of 0.5 equiv of aldehyde and 5-aminopyrazol afforded the corresponding adduct in 94% yield (entry 7). results [19]. Considering this, it was found that the combination of DCE, and an excess of 0.5 equiv of aldehyde and 5-aminopyrazol afforded the corresponding adduct in 94% yield (entry 7). Once the reaction conditions were optimized, we examined the scope of this threecomponent reaction using different substituted aromatic, heteroaromatic and aliphatic aldehydes. The structures and yields of the obtained products are shown in Table 2.
A plausible mechanism for the formation of these embelin derivatives (4) is shown in Scheme 1. Knoevenagel condensation between the aldehyde (2) and embelin (1) results in the formation of a methylene quinone reactive intermediate (A), which is trapped by the 3-phenyl-5-aminopyrazole (3). The reaction takes place via Michael addition to the methylene quinone system by the nucleophilic carbon in the α position to the amino group of the 3-phenyl-5-aminopyrazole to give the intermediate (B) which after a proton transfer gives the dihydropyridine system by intramolecular cyclization and dehydration.
These compounds were subsequently subjected to cellular phenotypic assays against eight different tumor cell lines. Three hematologic tumor cell lines, HEL (acute erythroid leukemia), K-562 (chronic myeloid leukemia) and HL-60 (acute myeloid leukemia), and five breast cancer cell lines (SKBR3, MCF-7, MDA-MB-231, BT-549, HS-578T). The primate nonmalignant kidney Vero cell line was used as control of cytotoxicity. The results obtained are shown in Table 3.
None of the compounds evaluated showed significant cytotoxic activity in the triple negative breast cancer cell lines MDA-MB-231 and HS-578T. Several of the compounds showed good cytotoxic activity against the rest of cell lines evaluated with IC 50 values between 0.7 and 7.5 µM.
In general terms, the best results were obtained in the leukemia cell lines compared to the breast cancer cell lines. Concerning the leukemia cell lines, compound 4a, with a 4-NO 2 group, exhibited the best cytotoxic activity in HL60 with an IC 50 of 0.70 ± 0.14 µM. This compound also showed good activity values in HEL (1.05 ± 0.35 µM) and K562 (1.25 ± 0.35 µM). Derivatives 4c, 4d, 4e and 4g, with substituents 4-Cl, 4-Br, 4-F and 4-CF 3 , respectively, also presented good values of cytotoxic activity in the three leukemia cell lines with IC 50 values between 0.90 and 3.30 µM. The best IC 50 value in acute erythroid leukemia (HEL) was achieved with compound 4g (1.00 ± 0.42 µM), whereas in chronic Once the reaction conditions were optimized, we examined the scope of this three-component reaction using different substituted aromatic, heteroaromatic and aliphatic aldehydes. The structures and yields of the obtained products are shown in Table  2. Once the reaction conditions were optimized, we examined the scope of this three-component reaction using different substituted aromatic, heteroaromatic and aliphatic aldehydes. The structures and yields of the obtained products are shown in Table  2. Once the reaction conditions were optimized, we examined the scope of this three-component reaction using different substituted aromatic, heteroaromatic and aliphatic aldehydes. The structures and yields of the obtained products are shown in Table  2. Once the reaction conditions were optimized, we examined the scope of this three-component reaction using different substituted aromatic, heteroaromatic and aliphatic aldehydes. The structures and yields of the obtained products are shown in Table  2. Once the reaction conditions were optimized, we examined the scope of this three-component reaction using different substituted aromatic, heteroaromatic and aliphatic aldehydes. The structures and yields of the obtained products are shown in Table  2. Once the reaction conditions were optimized, we examined the scope of this three-component reaction using different substituted aromatic, heteroaromatic and aliphatic aldehydes. The structures and yields of the obtained products are shown in Table  2. Once the reaction conditions were optimized, we examined the scope of this three-component reaction using different substituted aromatic, heteroaromatic and aliphatic aldehydes. The structures and yields of the obtained products are shown in Table  2. As we can observe, different yields were obtained depending on the type of aldehyde used in the Knoevenagel condensation. Thus, with aromatic aldehydes substituted with both electron-withdrawing (4a-4k) and electron-donating groups (4l-4o) good yields (73-98%) were obtained. The use of heteroaromatic aldehydes led to the corresponding products (4p-4s) in moderate yields (42-63%). This methodology is also tolerant to aliphatic aldehydes since the corresponding dihydro-1H-pyrazolo[1, 3-b]pyridine As we can observe, different yields were obtained depending on the type of aldehyde used in the Knoevenagel condensation. Thus, with aromatic aldehydes substituted with both electron-withdrawing (4a-4k) and electron-donating groups (4l-4o) good yields (73-98%) were obtained. The use of heteroaromatic aldehydes led to the corresponding products (4p-4s) in moderate yields (42-63%). This methodology is also tolerant to aliphatic aldehydes since the corresponding dihydro-1H-pyrazolo[1, 3-b]pyridine As we can observe, different yields were obtained depending on the type of aldehyde used in the Knoevenagel condensation. Thus, with aromatic aldehydes substituted with both electron-withdrawing (4a-4k) and electron-donating groups (4l-4o) good yields (73-98%) were obtained. The use of heteroaromatic aldehydes led to the corresponding products (4p-4s) in moderate yields (42-63%). This methodology is also tolerant to aliphatic aldehydes since the corresponding dihydro-1H-pyrazolo[1, 3-b]pyridine As we can observe, different yields were obtained depending on the type of aldehyde used in the Knoevenagel condensation. Thus, with aromatic aldehydes substituted with both electron-withdrawing (4a-4k) and electron-donating groups (4l-4o) good yields (73-98%) were obtained. The use of heteroaromatic aldehydes led to the corresponding products (4p-4s) in moderate yields (42-63%). This methodology is also tolerant to aliphatic aldehydes since the corresponding dihydro-1H-pyrazolo[1, 3-b]pyridine As we can observe, different yields were obtained depending on the type of aldehyde used in the Knoevenagel condensation. Thus, with aromatic aldehydes substituted with both electron-withdrawing (4a-4k) and electron-donating groups (4l-4o) good yields (73-98%) were obtained. The use of heteroaromatic aldehydes led to the corresponding products (4p-4s) in moderate yields (42-63%). This methodology is also toler- As we can observe, different yields were obtained depending on the type of aldehyde used in the Knoevenagel condensation. Thus, with aromatic aldehydes substituted with both electron-withdrawing (4a-4k) and electron-donating groups (4l-4o) good yields (73-98%) were obtained. The use of heteroaromatic aldehydes led to the corresponding products (4p-4s) in moderate yields (42-63%). This methodology is also toler- As we can observe, different yields were obtained depending on the type of aldehyde used in the Knoevenagel condensation. Thus, with aromatic aldehydes substituted with both electron-withdrawing (4a-4k) and electron-donating groups (4l-4o) good yields (73-98%) were obtained. The use of heteroaromatic aldehydes led to the corresponding products (4p-4s) in moderate yields (42-63%). This methodology is also toler- As we can observe, different yields were obtained depending on the type of aldehyde used in the Knoevenagel condensation. Thus, with aromatic aldehydes substituted with both electron-withdrawing (4a-4k) and electron-donating groups (4l-4o) good yields (73-98%) were obtained. The use of heteroaromatic aldehydes led to the corresponding products (4p-4s) in moderate yields (42-63%). This methodology is also toler- As we can observe, different yields were obtained depending on the type of aldehyde used in the Knoevenagel condensation. Thus, with aromatic aldehydes substituted with both electron-withdrawing (4a-4k) and electron-donating groups (4l-4o) good yields (73-98%) were obtained. The use of heteroaromatic aldehydes led to the corresponding products (4p-4s) in moderate yields (42-63%). This methodology is also toler- As we can observe, different yields were obtained depending on the type of aldehyde used in the Knoevenagel condensation. Thus, with aromatic aldehydes substituted with both electron-withdrawing (4a-4k) and electron-donating groups (4l-4o) good yields (73-98%) were obtained. The use of heteroaromatic aldehydes led to the corresponding products (4p-4s) in moderate yields (42-63%). This methodology is also toler- As we can observe, different yields were obtained depending on the type of aldehyde used in the Knoevenagel condensation. Thus, with aromatic aldehydes substituted with both electron-withdrawing (4a-4k) and electron-donating groups (4l-4o) good yields (73-98%) were obtained. The use of heteroaromatic aldehydes led to the corresponding products (4p-4s) in moderate yields (42-63%). This methodology is also toler- As we can observe, different yields were obtained depending on the type of aldehyde used in the Knoevenagel condensation. Thus, with aromatic aldehydes substituted with both electron-withdrawing (4a-4k) and electron-donating groups (4l-4o) good yields (73-98%) were obtained. The use of heteroaromatic aldehydes led to the corresponding products (4p-4s) in moderate yields (42-63%). This methodology is also toler- As we can observe, different yields were obtained depending on the type of aldehyde used in the Knoevenagel condensation. Thus, with aromatic aldehydes substituted with both electron-withdrawing (4a-4k) and electron-donating groups (4l-4o) good yields (73-98%) were obtained. The use of heteroaromatic aldehydes led to the corresponding products (4p-4s) in moderate yields (42-63%). This methodology is also toler- As we can observe, different yields were obtained depending on the type of aldehyde used in the Knoevenagel condensation. Thus, with aromatic aldehydes substituted with both electron-withdrawing (4a-4k) and electron-donating groups (4l-4o) good yields (73-98%) were obtained. The use of heteroaromatic aldehydes led to the corresponding products (4p-4s) in moderate yields (42-63%). This methodology is also toler- As we can observe, different yields were obtained depending on the type of aldehyde used in the Knoevenagel condensation. Thus, with aromatic aldehydes substituted with both electron-withdrawing (4a-4k) and electron-donating groups (4l-4o) good yields (73-98%) were obtained. The use of heteroaromatic aldehydes led to the corresponding products (4p-4s) in moderate yields (42-63%). This methodology is also toler-a Isolated yields. These compounds were subsequently subjected to cellular phenotypic assays against eight different tumor cell lines. Three hematologic tumor cell lines, HEL (acute erythroid leukemia), K-562 (chronic myeloid leukemia) and HL-60 (acute myeloid leukemia), and five breast cancer cell lines (SKBR3, MCF-7, MDA-MB-231, BT-549, HS-578T). The primate non-malignant kidney Vero cell line was used as control of cytotoxicity. The results obtained are shown in Table 3.  According to these first results, the importance of substituents in para position of the phenyl ring at the 1,4-dihydropyridine ring, is highlighted. For the derivatives 4f, and 4h, having a -F and a -NO 2 group, at the meta position a loss of cytotoxic activity was detected compared to the corresponding para-substituted derivatives (4e and 4a). Interestingly, the replacement of these electron-withdrawing groups at the para position of the phenyl ring by others such as the -COOH (4i), or -CO 2 CH 3 (4j) also leads to a loss of activity.
On the other hand, the presence of electron-donor groups at the phenyl ring such as -NMe 2 , -3F-4MeO, 3-4-MeO, 3,4-methylenedioxy (4l, 4m, 4n and 4o) leads to worse values of IC 50 in the leukemia cell lines, compared with those of 4b (-Ph). A clear loss of activity when heteroaromatic rings were attached at the dihydropyridin nucleus (4p-4s) was also observed, while aliphatic substituents (4u, 4v, 4w) leads to higher values of IC 50 with the exception of the derivative 4t, with a cyclohexyl group, which keeps a good cytotoxic activity (1.30 ± 0.28 µM) against HEL.
All previously synthesized nitrogenated embelin derivatives were also evaluated in the Vero cell line of primate kidney, to determine its cytotoxicity in a non-tumor cell line and to study its viability for carrying out future in vivo tests. Interestingly, inhibition of cell viability induced by several compounds were 8-to 20-fold lower in Vero cells compared to hematological (e.g., 4c, 4d, 4g, 4t) and breast cancer (e.g., 4c, 4d, 4g) cell lines. Taking advantage of the versatility of this multicomponent reaction, and given the good results of cytotoxic activity obtained for some of the dihydro-1H-pyrazolo [3,4-b]pyridine derivatives previously synthesized, we decided to carry out modifications in the quinone and aminopyrazole components to evaluate the effect of these structural variations on the cytotoxic activity.
Several of the compounds evaluated exhibit good values of cytotoxic activity in the different cell lines studied. The derivative 4g shows a better selectivity against hematological tumor cell lines, a good cytotoxic activity in erythroleukemia (HEL, 1.00 ± 0.42 µM) and the presence of fluorinated groups in biologically active molecules, such as the trifluoromethyl group, is of great interest [20] due to metabolic degradation resistance [21]. Furthermore, 4g showed low cytotoxicity (IC 50 > 25 µM) in normal Vero cell line with a calculated selectivity index higher than 25-fold.
Taking into account all data mentioned above, compound 4g was selected to continue with the preparation of analogues with the -4-CF 3 Ph group retained.
As we can observe in Table 5, the shortening of the side chain leads to a loss of activity, except in the SKBR3 cell line, where compound 10 (R 2 = (CH 2 ) 5 CH 3 ) showed an increased antiproliferative activity (IC 50 = 1.50 ± 1.27 µM) with respect to derivative 4g. Derivatives 9-12 were also inactive against MDA-MB-231 and HS-578T cell lines.
The derivative 13 (Scheme 3) was also prepared using trimethyl silyl diazomethane in ether/methanol in order to evaluate the role of the free hydroxyl group on the cytotoxic activity. Compound 13 showed an IC 50 value > 10 µM in all cell lines evaluated, which confirms the importance of the hydroxyl group for the cytotoxic activity. Pharmaceuticals 2021, 14, 1026 7 shown in Scheme 2 [22]. Table 4 shows the yields obtained in the preparation of de tives 9-12.
The different substituted 3-phenyl-5-aminopyrazoles were synthesized from the corresponding β-ketonitriles and hydrazine in EtOH at reflux [23]. On the other hand, the β-ketonitriles could be easily obtained by modifying the procedure described by Liu et al.
The corresponding derivatives were obtained in good yields, from 76 to 94%, regardless of the nature of the substituent at the pyrazole ring (14a-14f). Furthermore, as we can see in Table 6, this methodology also allows us to use aminopyrazoles substituted with heteroaromatic groups such as derivative 14g (82%) having a 2-furyl group, or aliphatic groups such as 5-methyl-3-aminopyrazole (14h, 78%).
Some of the new derivatives showed good cytotoxic activities, especially in breast cancer cell lines. The derivative 14c, with a 4-F-Ph moiety at the pyrazole ring, showed the best cytotoxic activity with an IC 50 of 0.59 ± 0.00 µM in MCF7, the derivatives 14e with a 4-OMe substituent and 14g with a 2-furyl group also presented an IC 50 of 0.85 ± 0.03 µM and 0.93 ± 0.74 µM in the same cell line. Regarding the BT549 cell line, the presence of different substituents in the aromatic ring of the pyrazole seems to have a negative effect on the activity. For the SKBR3 cell line, several of the derivatives such as 14c (4-F-Ph), 14d (3-F-Ph), 14e (4-MeO-Ph) and 14g (2-furyl) presented a significant improvement in their cytotoxic activity with values of IC 50 from 2.01 ± 1.12 µM to 2.40 ± 0.28 µM. All of them also showed an IC 50 > 10 µM in MDA-MB-231 and HS-578BT cell lines.
The modified derivatives were evaluated against the eight tumor cell lines (Table 7). The corresponding derivatives were obtained in good yields, from 76 to 94%, regardless of the nature of the substituent at the pyrazole ring (14a-14f). Furthermore, as we can see in Table 6, this methodology also allows us to use aminopyrazoles substituted with heteroaromatic groups such as derivative 14g (82%) having a 2-furyl group, or aliphatic groups such as 5-methyl-3-aminopyrazole (14h, 78%).
The modified derivatives were evaluated against the eight tumor cell lines (Table 7).  The corresponding derivatives were obtained in good yields, from 76 to 94%, regardless of the nature of the substituent at the pyrazole ring (14a-14f). Furthermore, as we can see in Table 6, this methodology also allows us to use aminopyrazoles substituted with heteroaromatic groups such as derivative 14g (82%) having a 2-furyl group, or aliphatic groups such as 5-methyl-3-aminopyrazole (14h, 78%).
The modified derivatives were evaluated against the eight tumor cell lines (Table 7). The corresponding derivatives were obtained in good yields, from 76 to 94%, regardless of the nature of the substituent at the pyrazole ring (14a-14f). Furthermore, as we can see in Table 6, this methodology also allows us to use aminopyrazoles substituted with heteroaromatic groups such as derivative 14g (82%) having a 2-furyl group, or aliphatic groups such as 5-methyl-3-aminopyrazole (14h, 78%).
The modified derivatives were evaluated against the eight tumor cell lines (Table 7). The corresponding derivatives were obtained in good yields, from 76 to 94%, regardless of the nature of the substituent at the pyrazole ring (14a-14f). Furthermore, as we can see in Table 6, this methodology also allows us to use aminopyrazoles substituted with heteroaromatic groups such as derivative 14g (82%) having a 2-furyl group, or aliphatic groups such as 5-methyl-3-aminopyrazole (14h, 78%).
The modified derivatives were evaluated against the eight tumor cell lines (Table 7).  The corresponding derivatives were obtained in good yields, from 76 to 94%, regardless of the nature of the substituent at the pyrazole ring (14a-14f). Furthermore, as we can see in Table 6, this methodology also allows us to use aminopyrazoles substituted with heteroaromatic groups such as derivative 14g (82%) having a 2-furyl group, or aliphatic groups such as 5-methyl-3-aminopyrazole (14h, 78%).
The modified derivatives were evaluated against the eight tumor cell lines (Table 7). The corresponding derivatives were obtained in good yields, from 76 to 94%, regardless of the nature of the substituent at the pyrazole ring (14a-14f). Furthermore, as we can see in Table 6, this methodology also allows us to use aminopyrazoles substituted with heteroaromatic groups such as derivative 14g (82%) having a 2-furyl group, or aliphatic groups such as 5-methyl-3-aminopyrazole (14h, 78%).
The modified derivatives were evaluated against the eight tumor cell lines ( The corresponding derivatives were obtained in good yields, from 76 to 94%, regardless of the nature of the substituent at the pyrazole ring (14a-14f). Furthermore, as we can see in Table 6, this methodology also allows us to use aminopyrazoles substituted with heteroaromatic groups such as derivative 14g (82%) having a 2-furyl group, or aliphatic groups such as 5-methyl-3-aminopyrazole (14h, 78%).
The modified derivatives were evaluated against the eight tumor cell lines ( The corresponding derivatives were obtained in good yields, from 76 to 94%, regardless of the nature of the substituent at the pyrazole ring (14a-14f). Furthermore, as we can see in Table 6, this methodology also allows us to use aminopyrazoles substituted with heteroaromatic groups such as derivative 14g (82%) having a 2-furyl group, or aliphatic groups such as 5-methyl-3-aminopyrazole (14h, 78%).
The modified derivatives were evaluated against the eight tumor cell lines (Table 7). Concerning the hematological tumor cell lines, we can observe an improvement in the cytotoxic activity for the derivative 14d (3-F-Ph) with an IC 50 of 1.05 ± 0.64 µM in HL60 cell line and 0.95 ± 0.4 µM in K562 cell line. Derivatives 14e (4-MeO-Ph) and 14g (2-furyl), as in some of the breast cancer cell lines, also have good cytotoxicity values in HL60 with IC 50 of 1.10 ± 0.14 and 1.10 ± 0.04 µM, respectively.
Next, we decided to evaluate the effect of the planarity of this type of structure on cytotoxic activity. Thus, when compound 4g was treated with DDQ, the corresponding pyrazolopyridin derivative (15) was obtained in 82% yield (Scheme 5), and it presented IC 50 values higher than 10 µM in all cell lines studied.
Taking into account all mentioned results, Figure 2 displays a summary of the established structure-activity relationships (SARs) for these new antiproliferative dihydro-1Hpyrazolo [1,3-b]pyridine embelin derivatives.
Since other embelin derivatives have shown inhibitory activity against the human protein kinase CK2 [17,18], we think that CK2 could be also the target of this type of compounds. In this sense, docking studies were carried out with the most active compounds using Glide software [25] on the reported crystal structure of human protein kinase CK2 alpha subunit in complex with the inhibitor CX-4945 (PDB 3PE1). An analysis of the docking results showed that the compounds fit well and, as shown in Figure 3, the active site is fully occupied by the compound 4g, the aliphatic chain was located at the bottom of the pocket. In this case, the alkyl chain established hydrophobic interactions with the residues Phe 113, Ile 95, Val 66 and Lys 68. Furthermore, two hydrogen bond interactions in the hinge region were observed between the residue Val 116 and the NH of the dihydropyridine ring and one of the quinonic carbonyl which explains the good value of docking score obtained during the simulation (−8.430 Kcal/mol). the cytotoxic activity for the derivative 14d (3-F-Ph) with an IC50 of 1.05 ± 0.64 HL60 cell line and 0.95 ± 0.4 μM in K562 cell line. Derivatives 14e (4-MeO-Ph) (2-furyl), as in some of the breast cancer cell lines, also have good cytotoxicity v HL60 with IC50 of 1.10 ± 0.14 and 1.10 ± 0.04 μM, respectively.
Next, we decided to evaluate the effect of the planarity of this type of struc cytotoxic activity. Thus, when compound 4g was treated with DDQ, the corresp pyrazolopyridin derivative (15) was obtained in 82% yield (Scheme 5), and it pr IC50 values higher than 10 μM in all cell lines studied. Taking into account all mentioned results, Figure 2 displays a summary o tablished structure-activity relationships (SARs) for these new antiproliferativ dro-1H-pyrazolo [1,3-b]pyridine embelin derivatives. Since other embelin derivatives have shown inhibitory activity against the protein kinase CK2 [17,18], we think that CK2 could be also the target of this compounds. In this sense, docking studies were carried out with the most acti pounds using Glide software [25] on the reported crystal structure of human pro nase CK2 alpha subunit in complex with the inhibitor CX-4945 (PDB 3PE1). An an the docking results showed that the compounds fit well and, as shown in Figur active site is fully occupied by the compound 4g, the aliphatic chain was locate Scheme 5. Synthesis of derivative 15 from 4g. Table 7. Cytotoxic activity of derivatives 14a-14h in human tumor cell lines (leukemia and breast) and in primate non-tumor kidney Vero cells a .
The corresponding derivatives were obtained in good yields, from 76 to 94%, regardless of the nature of the substituent at the pyrazole ring (14a-14f). Furthermore, as we can see in Table 6, this methodology also allows us to use aminopyrazoles substituted with heteroaromatic groups such as derivative 14g (82%) having a 2-furyl group, or aliphatic groups such as 5-methyl-3-aminopyrazole (14h, 78%).
The modified derivatives were evaluated against the eight tumor cell lines (Table 7). Understanding the physicochemical properties and the pharmacokinetic profile (absorption, distribution, metabolism and excretion) of molecules is an essential step to avoid failures in the selection of lead molecules during the different stages of development and discovery of new drugs. Therefore, it is essential to design chemical leads with acceptable ADME and good drug-like properties. However, about the drug-likeness of natural product derivatives, it is important to mention that many derivatives of them do not comply with drug-like rules during the lead optimization process. A lot of these compounds are found outside the drug-like space because they usually have a higher molecular weight and can be more complex than drug-like. Although the greatest value of the use of the privileged structures present in natural products is that they were optimized during the course of evolution and also, they can explore parts of chemical space that synthetic drug-like compounds do not essentially cover. Taking into account all mentioned results, Figure 2 displays a sum tablished structure-activity relationships (SARs) for these new antipr dro-1H-pyrazolo [1,3-b]pyridine embelin derivatives. Since other embelin derivatives have shown inhibitory activity ag protein kinase CK2 [17,18], we think that CK2 could be also the targe compounds. In this sense, docking studies were carried out with the m pounds using Glide software [25] on the reported crystal structure of h nase CK2 alpha subunit in complex with the inhibitor CX-4945 (PDB 3PE the docking results showed that the compounds fit well and, as shown active site is fully occupied by the compound 4g, the aliphatic chain w  Understanding the physicochemical properties and the pharmacokinetic profile (absorption, distribution, metabolism and excretion) of molecules is an essential step to avoid failures in the selection of lead molecules during the different stages of development and discovery of new drugs. Therefore, it is essential to design chemical leads with acceptable ADME and good drug-like properties. However, about the drug-likeness of natural product derivatives, it is important to mention that many derivatives of them do not comply with drug-like rules during the lead optimization process. A lot of these compounds are found outside the drug-like space because they usually have a higher molecular weight and can be more complex than drug-like. Although the greatest value of the use of the privileged structures present in natural products is that they were optimized during the course of evolution and also, they can explore parts of chemical space that synthetic drug-like compounds do not essentially cover.
In this sense in silico prediction of physicochemical properties, ADME parameters, Lipinski's rule of five (Ro5), and Jorgensen's rule of three and drug-likeness were determined for dihydro-1H-pyrazolo[1,3-b]pyridine embelin derivatives with the best an- In this sense in silico prediction of physicochemical properties, ADME parameters, Lipinski's rule of five (Ro5), and Jorgensen's rule of three and drug-likeness were determined for dihydro-1H-pyrazolo [1,3-b]pyridine embelin derivatives with the best antiproliferative values (4a, 4c, 4e, 4g, 14c, 14d, 14e and 14g). This study was performed using the Qikprop module of Schrödinger software [25], which predicts physically and pharmaceutically relevant properties of organic molecules on the full 3D molecular structure and provides range to compare with the molecular properties of known 95% drugs. The predicted parameters and their recommended values are presented in Table 8. The rule of five [26] is a widely used filter to indicate if a compound presents good oral bioavailability. However, other parameters such as the total number of rotatable bonds and the polar surface area (PSA) are found to be important predictors of good oral absorption and bioavailability, independent of molecular weight [27].  #rotor  12  11  11  11  11  11  12  11  0 to 15   donorHB  3  3  3  3  3  3  3  3  As expected, the results presented in Table 8 show that the selected compounds do not meet all the properties required by the rules, such as having a MW < 500, log P < 5, thus showing two violations for Lipinski's rule and a violation of the rule of three is established because the values of log S are <−5.7. Therefore, according the rules of three and five, embelin derivatives have higher lipophilicity and lower solubility in water, this result is reasonable due to the presence of the 11-carbon aliphatic chain of the embelin moiety. However, for an orally active compound, two violations of Lipinski's rule and one violation of the rule of three are acceptable. Thus, these compounds show their properties having the maximum number of infractions of these rules.
Most of the molecules showed excellent predicted blood/brain partition co-efficient (QPlogBB) values which is a measure of the ability of a drug to cross the blood-brain barrier and also the blood-brain barrier mimic MDCK cell permeability (QPPMDCK) show satisfactory predictions for all the compounds. The calculated theoretical PSA values for all compounds allow to support the low ability of the compounds to penetrate the blood-brain barrier with the exception of compound 4a. The most widely recommended PSA cutoff values are about 120-140 Å. all molecules showed excellent predicted Caco-2 cell (model for the gut-blood barrier) permeability.
On the other hand, the prediction of oral drug absorption (percent human oral absorption) was highly satisfactory for all the compounds, moreover human serum albumin binding co-efficient (QPlogKhsa) were found to be within an acceptable range which in- dicates their strong binding with plasma protein with the exception of compounds 4g, 14d and 14e. Human serum albumin binding co-efficient is one of the key factors since it is related to the transport of many of the drugs to its targets once they enter into the circulatory system.
The presence of reactive functional groups (#rtvFG) is an important predicted additional property that alerts us to the presence of different groups such as silicon, aluminum, diazo, azide, carbonate, anhydride, etc., which is related to decomposition, reactivity and toxicity in vivo and also the presence of these groups can lead to false positives when the compounds are evaluated biologically while the predicted skin permeability (logKp) is within the recommended values.
Finally, the ADME profile of our compounds showed good values, mainly in those properties related to well calculated permeability, low toxicity and good protein-plasma interaction. These predictions linked to the values of biological activity are promising and, therefore, deserve further investigation for further optimization of the synthesized compounds.

General Experimental Procedures
IR spectra were obtained using a Fourier transform infrared spectrometer. NMR spectra were recorded in CDCl 3 or DMSO-d 6 at 500 or 600 MHz for 1 H NMR and 125 or 150 MHz for 13 C NMR. Chemical shifts are given in (δ) parts per million and coupling constants (J) in hertz (Hz). 1 H and 13 C spectra were referenced using the solvent signal as internal standard. Melting points were taken on a capillary melting point apparatus and are uncorrected. HREIMS were recorded using a high-resolution magnetic trisector (EBE) mass analyzer. Analytical thin-layer chromatography plates Polygram-Sil G/UV254 were used. Preparative thin-layer chromatography was carried out with Analtech silica gel GF plates (20 × 20 cm, 1000 Microns) using appropriate mixtures of ethyl acetate and hexanes. Microwave reactions were conducted in sealed glass vessels (capacity 5 mL) using a Biotage initiator microwave reactor. All solvents and reagents were purified by standard techniques reported [28] or used as supplied from commercial sources. All compounds were named using the ACD40 Name-Pro program, which is based on IUPAC rules. The embelin (1) used in the reactions was obtained from Oxalis erythrorhiza Gillies ex Hook and Arn following the procedure described in reference [29].

General Procedure for the Synthesis of Pyrazolo[3,4-b] Quinolin-5,8(4H,9H)-Dione Derivatives
To a MW tube equipped with a magnetic stir bar, embelin, 1.5 equiv of aldehyde, 1.5 equiv of 3-amino-5-phenylpyrazole and 10 mol % EDDA in 2 mL of DCE were added. The MW tube was sealed, and the reaction mixture was irradiated at 150 • C for 10 min. The products were isolated by filtration or purified by Shepadex LH-20.  24.4 mg of 3-amino-5-phenylpyrazole (0.15 mmol) were dissolved in 2 mL of DCE and treated with 1.8 mg of EDDA (10 mol %). The tube was sealed, and the reaction mixture was irradiated at 150 • C for 10 min. Then, the reaction mixture was filtered and the obtained solid was washed with n-hex to yield 53.7 mg (89%) of 4g as an amorphous violet solid. Mp: 213.9-214.9 • C; 1   Following the general procedure described above, in a 5 mL MW tube, 35 mg of embelin (0.12 mmol), 27 mg of 4-dimethylaminobenzaldehyde (0.18 mmol) and 28.7 mg of 3-amino-5-phenylpyrazole (0.18 mmol) were dissolved in 2 mL of DCE and treated with 3.4 mg of EDDA (10 mol %). The tube was sealed, and the reaction mixture was irradiated at 150 • C for 10 min. Then, the reaction mixture was filtered and the obtained solid was washed with n-hex to yield 59.9 mg (88%) of 4l as an amorphous violet solid.