Synthesis, Docking Study and Kinase Inhibitory Activity of a Number of New Substituted Pyrazolo[3,4-c]pyridines

Abstract

A series of new pyrazolo[3,4-c]pyridines bearing various 1, 3, 5 or 1, 3, 7 pattern substitutions, were designed and synthesized. Some of them showed interesting inhibitory activity mainly against glycogen synthase kinase 3 (GSK3)α/β as well as against cdc2-like kinases 1 (CLK1) and dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), with good selectivity and remarkable structure–activity relationships (SARs), without being cytotoxic. Molecular simulations in correlation with biological data revealed the importance of the existence of N1-H as well as the absence of a bulky 7-substituent.

Neurodegenerative diseases are among the most challenging diseases with poorly known mechanism and lack of complete cure. Alzheimer’s disease (AD) in particular, is the most prevalent cause of dementia, very devastating for the patients and their families, provided that current treatments offer only modest symptomatic relief. This severe mental disorder is mostly aging-associated and represents a global health problem, since it currently affects more than 30 million people worldwide, and its incidence is predicted to rise significantly, due to the increasing average life span.¹⁾ In an effort to explain the pathogenesis of AD, many hypotheses have been explored, among them neurotransmitter modulation, chronic inflammation, metal-induced oxidative stress, elevated cholesterol, and glycogen synthase kinase 3 (GSK-3) implication are of major importance.²⁾ GSK-3 is a ubiquitous serine/threonine kinase, which was first described as the major regulator of glycogen metabolism. It was revealed that GSK-3 plays central roles in important cell signaling pathways, and its malfunction is associated with neurodegeneration and the pathogenesis of several diseases, including diabetes type II, immune and bipolar disorders, chronic inflammation, heart failure and cancer.³⁾ In AD, GSK-3 promotes neuronal death and is a linker between the two histopathological hallmarks: the deposition of extracellular senile plaques composed of amyloid-beta (Aβ) peptide, and the accumulation of hyperphosphorylated microtubule-binding tau protein, leading to tau aggregation and the formation of intracellular neurofibrillary tangles.⁴⁾ On the other hand, substantial evidence exists to support the involvement of additional phylogenetically and physiologically relevant protein kinases in neurodegenerative disorders, and, most notably, cyclin dependent kinase 5 (CDK5),⁵⁾ dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK 1A),⁶⁾ casein kinase 1 (CK1)⁷⁾ and cdc2-like kinases CLK1 and CLK2⁸⁾ are also investigated as potential targets for the development of novel AD therapeutics. Consequently, the exploitation of small molecule inhibitors for potential inhibitory activity against a set of the above mentioned protein kinases could offer interesting information and assist in the understanding of AD and related taupathies.

A great number of GSK3 inhibitors have been reported in the literature and some of them were found to ameliorate cognitive impairments⁹⁾ and reduce the amount of Aβ, together with the tau hyperphosphorylation in neuronal cells.¹⁰⁾ Although a number of putative allosteric inhibitors appeared in the literature,¹¹⁾ most of the reported GSK3 inhibitors are ATP competitive, and are often characterized by the presense of a central purine-like heterocyclic scaffold, substituted with a characteristic carboxamide residue.^12,13) Within this context and as part of a research program aimed to study modified purine analogues as potential protein kinase inhibitors, we have explored the pyrazolopyridine scaffold and present in this study the design, synthesis and evaluation of a number of new substituted pyrazolo[3,4-c]pyridines for inhibitory potency against a panel of eight protein kinases.

Results and Discussion

Chemistry

For the preparation of the target compounds we have used both nitroderivatives of 2-aminopicoline which were synthesized according to reported procedures.¹⁴⁾ 2-Amino-5-nitropicoline (1, Chart 1) was diazotized and the resulting pyridinone 2¹⁵⁾ was treatad with phosphorus oxychloride and provided the chloride 3.¹⁶⁾ This chloride was reduced using tin chloride and the resulting aminoderivative 4 was acetylated to give the acetamide 5.¹⁷⁾ Compound 5 was treated with isoamylnitrite in the presence of potassium acetate and acetic anhydride and this reaction furnished a mixture of the corresponding 1- and 2-acetylpyrazolo[3,4-c]pyridines, through a rearrangement of the intermediate N-nitroso compound.¹⁸⁾ The acetamides were not isolated but the acetyl group was cleaved upon treatment with methanolic ammonia to result in the pyrazolopyridine 6.^17,19) The heterocyclic compound 6 was then nitrated and the resulting 3-nitroderivative 7 was treated with methyl iodide, or 4-methoxybenzyl chloride in the presence of potassium carbonate, to provide both regio-isomers 8a, b and 9a, b, respectively, which were chromatographically separated and identified by means of two-dimensional (2D)-NMR experiments. More specifically, in the corresponding nuclear Overhauser effect (NOE) spectra we observed clear cross-peak correlation between the N1 methyl group of 8a, or the N1 methylene group of 8b with H-7, verifying unambiguously the N1 substitution. This correlation was, as expected not detected in the case of compounds 9a and b. The chlorine atom of compounds 8a and b was subsequently displaced upon treatment with cyclohexylamine or aniline to result in nitroderivatives 10a–d. This reaction was effected through a straightforward aromatic nucleophilic substitution when cyclohexylamine was used, however in the case of the weak nucleophile, aniline, the use of Buchwald–Hartwig coupling conditions was nessecary.²⁰⁾ Compounds 10a–d were then catalytically reduced to the 3-aminoderivatives 11a–d and subsequently converted to the corresponding chloracetamides 12a–d, which were used for the preparation of the target aminoderivatives 13a–l. The 4-methoxybenzyl group of the compounds 13g–l was then removed in acidic media and derivatives 14a–f were prepared as well.

Chart 1

Reagents and conditions: a) NaNO₂, H₂SO₄, H₂O; b) POCl₃, 110°C; c) SnCl₂·2H₂O, HCl(c.), 55°C; d) Ac₂O, CH₂Cl₂, r.t.; e) (1): AcOK, Ac₂O, isoamyl nitrite, benzene, reflux, (2): NH₃(g.), MeOH, r.t.; f) HNO₃, H₂SO₄, 95°C; g) (1): K₂CO₃, MeCN, 0°C, (2): CH₃I for 8a, 9a or 4-methoxybenzyl chloride for 8b, 9b, 50°C; h) cyclohexylamine, dimethyl sulfoxide (DMSO), 150°C for 10a or cyclohexylamine, DMSO, microwave irradiation, 60 W, 158°C for 10c or aniline, X-Phos, Pd₂dba₃, CsCO₃, toluene, reflux for 10b, d; i) Pd/C, H₂, EtOH, 55 psi, r.t. for 11a–c or Pd/C, Et₃SiH, MeOH, r.t for 11d; j) chloroacetylchloride, Et₃N, THF, −40°C for 12a, c or 0°C for 12b, d; k) dimethylamine (5.6 M solution in EtOH) for 13a, d, g, j or 1-methylpiperazine for 13b, e, h, k or aniline for 13c, f, i, l, EtOH, 80°C; l) trifluoroacetic acid, 70°C.

In order to synthesize the corresponding isomeric analogues we used 2-amino-3-nitropicoline (15, Chart 2), which by a reaction sequence analogous to the above mentioned reported, corresponding to the isomer 1, was converted to the regio-isomers 22a and 23, as well as 22b. Concerning the preparation of the 4-methoxybenzyl-substituted compounds, only the N1 isomer 22b was isolated, presumably due to the significant steric hindrance exerted from the 3- nitro group.

Chart 2

Reagents and conditions: a) NaNO₂, H₂SO₄, H₂O; b) POCl₃, 110°C; c) SnCl₂·2H₂O, HCl(c.), 55°C; d) Ac₂O, CH₂Cl₂, r.t.; e) (1): AcOK, Ac₂O, isoamyl nitrite, benzene, reflux, (2): NH₃(g_.), MeOH, r.t.; f) HNO₃, H₂SO₄, 95°C; g) (1): K₂CO₃, MeCN, 0°C, (2): CH₃I for 22a, 23 or 4-methoxybenzyl chloride for 22b, 50°C.

Compounds 22a and b were selected for the insertion of the 7-alkyl or arylamino substituent, consequently, they were treated with cyclohexylamine (Chart 3) and provided the 7-substituted nitroderivatives 24a and b, respectively. The nitro group of the former compounds was then reduced and the resulting amines 25a and 26a were chloracetylated to give the chloracetamides 25b and 26b, which were treated with suitable amines to result in the target derivatives 27a–c and 28 as well as to its unprotected analogue 29. On the other hand, 22a was treated with aniline, to provide compound 30. As previously reported the 7-chloropyrazolo[3,4-c]pyridine is susceptible to nucleophilic substitution, even by relatively weak arylamines.¹⁷⁾ The nitroderivative 30 was finaly converted in three steps to the target derivatives 33a–c.

Chart 3

Reagents and conditions: a) cyclohexylamine, DMSO, 150°C; b) Pd/C, H₂, EtOH, 55 psi, r.t.; c) chloroacetylchloride, Et₃N, THF, −40°C; d) dimethylamine (5.6 M solution in EtOH) for 27a, 28 or 1-methylpiperazine for 27b, or aniline for 27c, EtOH, 80°C; e) trifluoroacetic acid, 70°C; f) aniline, 2-ethoxyethanol, 130°C; g) dimethylamine (5.6 M solution in EtOH) for 33a, or 1-methylpiperazine for 33b, or aniline for 33c, EtOH, 80°C.

Our attempts to substitute the chlorine atom of 22b with aniline were unsuccessful, presumably due to steric hindrance provided by the bulky 1-benzyl group. Thus we used the nitroderivative 21 (Chart 4) which was easily substituted to provide the phenylamino analogue 34. This compound was treated with 4-methoxybenzylchloride to provide selectively the corresponding N2-isomer 35, which following a procedure analogous to the described above, provided the target compounds 38a, b and 39a, b.

Chart 4

Reagents and conditions: a) aniline, 1,4-dioxane, reflux; b) (1): K₂CO₃, MeCN, 0°C, (2): 4-methoxybenzyl chloride, 50°C; c) Pd/C, H₂, EtOH, 55 psi, r.t.; d) chloroacetylchloride, Et₃N, THF, 0°C; e) dimethylamine (5.6 M solution in EtOH) for 38a or 1-methylpiperazine for 38b, EtOH, 80°C; f) trifluoroacetic acid, 70°C.

Biological Evaluation

The kinase inhibitory activity of the new compounds was tested against a panel of protein kinases and the results concerning the derivatives found to possess IC₅₀ values <10 µM are presented in Table 1. 6-Bromoindirubin-3′-oxime (6-BIO), a selective inhibitor of GSK-3, was used as a reference compound.²¹⁾ It is remarkable to notice that even if the vast majority of the synthesized new compounds are not endowed with interesting inhibitory activity against most of the kinases tested, the substitution pattern around the central heterocyclic ring system plays a crucial role on the inhibitory activity when present. Note here that, the kinases affected in the tested panel are structurally related, as they are members of the same group of the kinome, the CMGC cluster (for CDK, mitogen-activated protein kinase (MAPK), GSK3 and CLK).

Table 1. IC₅₀ Values (µM) of the Most Active Compounds against Protein Kinases Results Are Averages of at Least Two Independent Experiments

Compd	Hs_CDK5/p25	Mm_CLK1	Rn_DYRK1A	Ssc_GSK3α/β	Ssc_CK1δ/ε	Hs_HASPIN	Hs_AURKB	Hs_RIPK3
14a	>10	>10	>10	1	>10	>10	>10	>10
14b	>10	1.3	4.5	>10	>10	>10	>10	>10
14c	3.4	7	>10	0.5	>10	>10	>10	>10
14d	>10	>10	>10	0.55	>10	>10	>10	>10
14e	>10	1.1	4	0.4	>10	>10	>10	>10
14f	>10	6	>10	1	>10	>10	>10	>10
6-BIO	0.14	ND	1.30	0.014	1.90	ND	ND	ND

In general, the 7-substituted pyrazolopyridines, possessing either a phenylamino- or a cyclohexylamino-group, did not show any activity regardless the decoration of the rest of the scaffold. On the contrary, the corresponding 5-substituted analogues presented quite interesting structure–activity relationships (SARs). Among this group, compounds bearing methyl or 4-methoxybenzyl substitution at position 1, were again found inactive, with the exception of 13d, which appear to demonstrate a slight inhibitory effect (IC₅₀: 2.8 µM) against CDK5. However, the majority of the corresponding 1-unsubstituted analogues 14a–f, have shown interesting activity and various degree of specificity. More precisely, compounds 14a, c and d, e inhibited GSK3α/β with IC₅₀ values within 0.4–1 µM and among them, 14a and d, both containing the 3-dimethylaminoacetylamino substitution are selective towards this kinase. Compounds 14b and e, bearing a 3-(4-methylpiperazin-1-yl)aminoacetylamino substitution, show inhibitory activity against CLK1 and DYRK1A with IC₅₀ values in the range of 1.1–4.5 µM, while 14e is simultaneously active against GSK3α/β as mentioned before, being the most active analogue in the series showing IC₅₀ value 0.4 µM. Compound 14c, which possesses an aromatic group at position 3 has shown moderate inhibition against CDK5 and CLK 1, while its 5-phenylamino-counterpart 14f, demonstrated also a slight activity against CLK1. As a remark, the presence of a 5-alkyl or aryl group although important, influence equally the activity, nevertheless the contribution of a pyrazole NH is crucial for inhibitory activity of this type of compounds, mainly against GSK3α/β and in a lesser extent against CLK1 and DYRK1A.

With the aim to estimate the potential antiproliferative activity of compounds 14a–f, we have also evaluated their cytotoxicity against four breast cancer cell lines, namely MCF7, T47D, MDA-MB-231 and HCC1954 (data not shown). All derivatives presented IC₅₀ values ≥10 µM, providing evidence that they do not possess remarkable cytotoxic properties.

The aforementioned findings could argue in favor of a potential use of this type of derivatives against neurodegenerative diseases and this possibility is currently under investigation.

Theoretical Calculations

To gain insight to the putative interaction mode between the active pyrazolopyridine derivatives 14a–f and GSK3α/β, docking calculations were performed by implementing the Glide SP algorithm and post-docking flexible minimization utilizing MacroModel.^22–26) The co-crystal structure of GSK3β with indirubin was used as a template for docking calculations (pdb. id: 1UV5).²¹⁾ The predicted binding mode of the pyrazolopyridine scaffold is depicted in Fig. 1. The inhibitors bind the active site of the kinase through a typical type-I interaction mode (Fig. 1A). Two hydrogen bonds are formed between the pyrazole nitrogens of positions 1 and 2 and the D133 carbonyl of the hinge region, while a third bond is formed between the exocyclic amide nitrogen of position 3 and the V135 backbone amide. An additional hydrogen bond between the nitrogen position 3-substituent and the sidechain of Y134 further stabilizes the complex (Fig. 1B). This interaction mode is in excellent agreement with the two basic SAR notions derived from experiment. Indeed, capping of N1 diminishes activity as one of the hydrogen bonds would be excluded while the geometry of the rest would be moderately perturbed, as evident for analogues 13a–f. Moreover, the lack of activity for analogues carrying a bulky group on position 7 can be explained by the existence of extensive steric clashes with the hinge backbone and the gatekeeper sidechain (for example, derivative 39a as compared to 14a and d as well as 39b to 14b and e). Within the series of active compounds 14a–f, the preference for a more flexible substitution in position 5 such as a cyclohexyl instead of a phenyl ring can be explained by the enhanced capacity of the aliphatic group to fit to the plasticity of the cavity entrance as defined by the glycine (Gly)-loop.

Fig. 1. A. The Predicted Type-I Binding Mode of the Pyrazolopyridine Analogue 14c in the Active Site of GSK3β Kinase, Depicted as an Electrostatic Potential-Coloured Molecular Surface; B. The Pyrazolopyridine 14c (Shown in a Ball-and-Stick Representation) Is Stabilized in the ATP-Binding Pocket of GSK3β by Four Hydrogen Bonds (Dashed Lines) Formed between the Inhibitor Heterocyclic or Exocyclic Nitrogens and the Enzyme

Conclusion

In conclusion we have designed and synthesized a number of new N-substituted (pyrazolo[3,4-c]pyridin-3-yl)acetamides, possessing a variety of substituents on the heterocyclic ring system, in order to study their kinase inhibitory profile. Among the derivatives, only compounds that bear 5-aryl or alkyl substituents and retain the pyrazole nitrogens unsubstituted, have shown inhibition of GSK3α/β, with adequate selectivity, without being cytotoxic. Docking calculations showed that the aforementioned combination of substituents can provide a rationale for the further development of this original versatile lead into highly potent and selective inhibitors of kinase GSK3α/β.

Experimental

Chemistry

Melting points were determined on a Büchi apparatus and are uncorrected. ¹H-NMR spectra and 2D spectra were recorded on a Bruker Avance III 600 or a Bruker Avance DRX 400 instrument, whereas ¹³C-NMR spectra were recorded on a Bruker Avance III 600 or a Bruker AC 200 spectrometer in deuterated solvents and were referenced to tetramethylsilane (TMS) (δ scale). The signals of ¹H and ¹³C spectra were unambiguously assigned by using 2D-NMR techniques: ¹H–¹H correlation spectroscopy (COSY), nuclear Overhauser effect spectroscopy (NOESY), hetero-nuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond connectivity (HMBC). Mass spectra were recorded with a LTQ Orbitrap Discovery instrument, possessing an Ionmax ionization source. Flash chromatography was performed on Merck silica gel 60 (0.040–0.063 mm). Analytical TLC was carried out on precoated (0.25 mm) Merck silica gel F-254 plates. The purity of all the synthesized compounds was >95% as ascertained by elemental analysis. Elemental analyses were undertaken using a PerkinElmer, Inc. PE 240C elemental analyzer (Norwalk, CT, U.S.A.) and the measured values for C, H, and N were within±0.4% of the theoretical values.

5-Chloro-3-nitro-1H-pyrazolo[3,4-c]pyridine (7)

Chloro derivative 6 (490 mg, 3.19 mmol) was dissolved into concentrated sulfuric acid (9.50 mL) at 0°C and then a mixture of concentrated sulfuric acid (0.49 mL) and nitric acid (0.49 mL) was added dropwise into this solution. This reaction mixture was heated at 95°C for 1h. Upon cooling, it was poured into crashed ice, neutralized with an aqueous solution of NH₃ and the product was extracted with ethyl acetate (3×100 mL). The combined organic layers were dried over Na₂SO₄ and evaporated under reduced pressure to provide 600 mg of pure 7 as a yellow solid. Yield 94%, mp: 195–197°C (EtOAc). ¹H-NMR (400 MHz, DMSO-d₆) δ: 8.10 (1H, d, H-4, J=1.0 Hz), 9.16 (1H, d, H-7, J=1.0 Hz). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 113.3 (C-4), 122.1 (C-3a), 137.1 (C-7), 137.8 (C-7a), 144.0 (C-3). Anal. Calcd for C₆H₃ClN₄O₂: C, 36.29; H, 1.52; N, 28.22. Found: C, 36.43; H, 1.61; N, 28.45.

General Procedure for the Synthesis of 1-Substituted Derivatives 8a, b and 2-Substituted Derivatives 9a, b

Potassium carbonate (530 mg, 3.84 mmol) was added into a solution of nitro derivative 7 (540 mg, 2.72 mmol) in anhydrous acetonitrile (20 mL) at 0°C and under argon, and this solution was stirred for 30 min, followed by dropwise addition of iodomethane (0.22 mL, 3.53 mmol) or 4-methoxybenzyl chloride (0.41 mL, 3 mmol). This reaction mixture was heated at 50°C for 5 h. Upon completion of the reaction, the solvent was evaporated and the residue was extracted with ethyl acetate (3×100 mL) and water (100 mL). The combined organic layers were dried over Na₂SO₄ and evaporated under reduced pressure. The crude product was then purified by column chromatography (silica gel) to provide pure compounds 8a, b and 9a, b.

5-Chloro-1-methyl-3-nitro-1H-pyrazolo[3,4-c]pyridine (8a) and 5-Chloro-2-methyl-3-nitro-2H-pyrazolo[3,4-c]pyridine (9a)

These compounds were prepared according to the general procedure described above. Purification was effected using a mixture of cyclohexane–ethyl acetate (7 : 3, v/v) as the eluent.

Data for 8a: Yield 80%. Pale yellow solid, mp: 172°C (Et₂O). ¹H-NMR (400 MHz, CDCl₃) δ: 4.34 (3H, s, CH₃), 8.16 (1H, d, H-4, J=1.0 Hz), 8.89 (1H, d, H-7, J=1.0 Hz). ¹³C-NMR (50 MHz, CDCl₃) δ: 38.0 (CH₃), 114.4 (C-4), 123.3 (C-3a), 134.2 (C-7), 137.5 (C-7a), 145.9 (C-3). Anal. Calcd for C₇H₅ClN₄O₂: C, 39.55; H, 2.37; N, 26.35. Found: C, 39.49; H, 2.47; N, 26.52.

Data for 9a: Yield 14%. Pale yellow solid, mp: 177°C (Et₂O). ¹H-NMR (400 MHz, CDCl₃) δ: 4.67 (3H, s, CH₃), 8.03 (1H, d, H-4, J=1.0 Hz), 9.13 (1H, d, H-7, J=1.0 Hz). ¹³C-NMR (50 MHz, CDCl₃) δ: 43.8 (CH₃), 112.7 (C-4), 122.6 (C-3a), 136.9 (C-7a), 141.5 (C-5), 144.8 (C-7), 147.3 (C-3). Anal. Calcd for C₇H₅ClN₄O₂: C, 39.55; H, 2.37; N, 26.35. Found: C, 39.66; H, 2.42; N, 26.14.

5-Chloro-1-(4-methoxybenzyl)-3-nitro-1H-pyrazolo[3,4-c]pyridine (8b) and 5-Chloro-2-(4-methoxybenzyl)-3-nitro-2H-pyrazolo[3,4-c]pyridine (9b)

These compounds were prepared according to the general procedure described above. Purification was effected using a mixture of cyclohexane–ethyl acetate–dichloromethane (9 : 0.5 : 0.5, v/v/v) as the eluent.

Data for 8b: Yield 80%. Yellow solid, mp: 185°C (EtOAc). ¹H-NMR (400 MHz, CDCl₃) δ: 3.79 (3H, s, OCH₃), 5.70 (2H, s, CH₂), 6.90 (2H, d, H-3′, H-5′, J=8.7 Hz), 7.30 (2H, d, H-2′, H-6′, J=8.7 Hz), 8.18 (1H, d, H-4, J=1.0 Hz), 8.67 (1H, d, H-7, J=1.0 Hz). ¹³C-NMR (50 MHz, CDCl₃) δ: 55.3 (OCH₃), 55.9 (CH₂), 114.5 (C-4), 114.8 (C-3′, C-5′), 123.8 (C-3a), 124.7 (C-1′), 129.5 (C-2′, C-6′), 134.8 (C-7), 136.9 (C-7a), 145.7 (C-3), 160.4 (C-4′). Anal. Calcd for C₁₄H₁₁ClN₄O₃: C, 52.76; H, 3.48; N, 17.58. Found: C, 52.95; H, 3.60; N, 17.33.

Data for 9b: Yield 7%. Yellow solid, mp: 157°C (EtOAc). ¹H-NMR (400 MHz, CDCl₃) δ: 3.75 (3H, s, OCH₃), 6.09 (2H, s, CH₂), 6.83 (2H, d, H-3′, H-5′, J=8.7 Hz), 7.36 (2H, d, H-2′, H-6′, J=8.7 Hz), 7.99 (1H, s, H-4), 9.16 (1H, s, H-7). ¹³C-NMR (50 MHz, CDCl₃) δ: 55.3 (OCH₃), 58.7 (CH₂), 112.9 (C-4), 114.4 (C-3′, C-5′), 122.9 (C-3a), 125.3 (C-1′), 130.1 (C-2′, C-6′), 136.9 (C-7a), 141.6 (C-5), 145.1 (C-7), 147.3 (C-3), 160.2 (C-4′). Anal. Calcd for C₁₄H₁₁ClN₄O₃: C, 52.76; H, 3.48; N, 17.58. Found: C, 53.01; H, 3.69; N, 17.44.

N-Cyclohexyl-1-methyl-3-nitro-1H-pyrazolo[3,4-c]pyridin-5-amine (10a)

Cyclohexylamine (1.14 mL, 10 mmol) was added under argon into a solution of the 5-chloro-pyrazolopyridine 8a (425 mg, 2 mmol) in anhydrous DMSO (3 mL) and this mixture was heated at 140°C for 14 h. Upon completion of the reaction, the mixture was diluted with water (100 mL) and extracted with dichloromethane (3×100 mL). The combined organic layers were washed with water (200 mL), then with a saturated aqueous solution of NaCl (200 mL) and finally were dried over Na₂SO₄ and evaporated under reduced pressure. The residue was purified by column chromatography (silica gel) using a mixture of cyclohexane–dichloromethane (1 : 9, v/v) as the eluent, to provide pure 10a (275 mg, yield 50%) as an orange colored solid. mp: 148–149°C (EtOAc). ¹H-NMR (400 MHz, CDCl₃) δ: 1.25–1.34 (3H, m, cyclohexyl H), 1.46 (2H, m, cyclohexyl H), 1.68 (1H, m, cyclohexyl H), 1.80 (2H, m, cyclohexyl H), 2.09 (2H, m, cyclohexyl H), 3.50 (1H, m, cyclohexyl H-1′), 4.22 (3H, s, CH₃), 5.24 (1H, br s, D₂O exch., NH), 6.96 (1H, s, H-4), 8.69 (1H, s, H-7). ¹³C-NMR (50 MHz, CDCl₃) δ: 24.8 (cyclohexyl C-3′, C-5′), 25.8 (cyclohexyl C-4′), 32.8 (cyclohexyl C-2′, C-6′), 38.0 (CH₃), 51.3 (cyclohexyl C-1′), 91.1 (C-4), 125.6 (C-3a), 132.4 (C-7), 133.8 (C-7a), 146.0 (C-3), 155.0 (C-5). Anal. Calcd for C₁₃H₁₇N₅O₂: C, 56.72; H, 6.22; N, 25.44. Found: C, 56.49; H, 6.02; N, 25.57.

1-Methyl-3-nitro-N-phenyl-1H-pyrazolo[3,4-c]pyridin-5-amine (10b)

Aniline (0.99 mL, 10.86 mmol) was added under argon to a solution of the 5-chloro-pyrazolopyridine 8a (900 mg, 4.24 mmol) in dry toluene (70 mL), followed by addition of cesium carbonate (7.3 g, 18.96 mmol), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (X-phos, 172 mg, 0.36 mmol) and tris(dibenzylideneacetone)dipalladium(0) (207 mg, 0.23 mmol) and the resulting mixture was refluxed for 20 h. Upon completion of the reaction, the mixture was filtered through a celite pad, the filtrate was vacuum-evaporated, extracted with ethyl acetate–water and the organic extracts were dried over Na₂SO₄ and evaporated to dryness. The crude product was purified by column chromatography (silica gel) using a mixture of dichloromethane–ethyl acetate (97 : 3, v/v) as the eluent, to provide pure compound 10b (670 mg, 75%) as a red solid. mp: 171–172°C (EtOAc). ¹H-NMR (400 MHz, DMSO-d₆) δ: 4.27 (3H, s, CH₃), 6.94 (1H, t, H-4′, J=7.2 Hz), 7.30 (2H, t, H-3′, H-5′, J=7.3 Hz), 7.38 (1H, d, H-4, J=1.0 Hz), 7.55 (2H, d, H-2′, H-6′, J=8 Hz), 9.07 (1H, d, H-7, J=1.0 Hz), 9.29 (1H, s, D₂O exch., NH). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 37.8 (CH₃), 93.9 (C-4), 118.2 (C-2′, C-6′), 120.9 (C-4′), 123.4 (C-3a), 128.7 (C-3′, C-5′), 134.3 (C-7a), 134.9 (C-7), 141.5 (C-1′), 144.9 (C-3), 152.9 (C-5). Anal. Calcd for C₁₃H₁₁N₅O₂: C, 57.99; H, 4.12; N, 26.01. Found: C, 58.22; H, 4.16; N, 25.73.

N-Cyclohexyl-1-(4-methoxybenzyl)-3-nitro-1H-pyrazolo[3,4-c]pyridin-5-amine (10c)

A mixture of compound 8b (2.50 g, 7.85 mmol) and cyclohexylamine (9 mL, 78.47 mmol) in dimethylsulfoxide (8 mL) was irradiated at 158°C (60 W, Milestone Start E) for 2 h. Upon cooling, the mixture was poured into water (300 mL) and extracted with dichloromethane (3×200 mL). The combined organic layers were washed with water (500 mL), then with a saturated aqueous solution of NaCl (400 mL) and finally were dried over Na₂SO₄ and evaporated under reduced pressure. The crude product was purified by column chromatography (silica gel) using a mixture of cyclohexane–ethyl acetate (1 : 1, v/v) as the eluent, to provide pure 10c (2 g, 66%) as an orange colored solid. mp: 125–126°C (CHCl₃/Et₂O). ¹H-NMR (400 MHz, CDCl₃) δ: 1.26 (3H, m, cyclohexyl H), 1.44 (2H, m, cyclohexyl H), 1.66 (1H, m, cyclohexyl H), 1.78 (2H, m, cyclohexyl H), 2.07 (2H, m, cyclohexyl H), 3.48 (1H, m, cyclohexyl H-1′), 3.79 (3H, s, OCH₃), 4.90 (1H, br s, D₂O exch., NH), 5.58 (2H, s, CH₂), 6.87 (2H, d, H-3″, H-5″, J=8.7 Hz), 6.91 (1H, d, H-4, J=1.0 Hz), 7.28 (2H, d, H-2″, H-6″, J=8.7 Hz), 8.40 (1H, d, H-7, J=1.0 Hz). ¹³C-NMR (50 MHz, CDCl₃) δ: 24.6 (cyclohexyl C-3′, C-5′), 25.7 (cyclohexyl C-4′), 32.7 (cyclohexyl C-2′, C-6′), 51.1 (cyclohexyl C-1′), 55.3 (OCH₃), 55.6 (CH₂), 91.0 (C-4), 114.7 (C-3″, C-5″), 125.6 (C-1″), 125.8 (C-3a), 129.4 (C-2″, C-6″), 132.8 (C-7a), 133.1 (C-7), 145.4 (C-3), 155.0 (C-5), 160.2 (C-4″). Anal. Calcd for C₂₀H₂₃N₅O₃: C, 62.98; H, 6.08; N, 18.36. Found: C, 63.22; H, 6.19; N, 18.09.

1-(4-Methoxybenzyl)-3-nitro-N-phenyl-1H-pyrazolo[3,4-c]pyridin-5-amine (10d)

This compound was prepared following a procedure analogous to that described for the synthesis of 10b, starting from 8b. The product was purified by column chromatography (silica gel) using a mixture of cyclohexane–ethyl acetate (7 : 3, v/v) as the eluent. Yield 68%. Red solid, mp: 179°C (EtOAc). ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.72 (3H, s, OCH₃), 5.79 (2H, s, CH₂), 6.92 (3H, m, H-3″, H-5″, H-4′), 7.29 (2H, t, H-3′, H-5′, J=7.5 Hz), 7.40 (3H, m, H-4, H-2″, H-6″), 7.54 (2H, d, H-2′, H-6′, J=7.5 Hz), 9.15 (1H, d, H-7, J=1.0 Hz), 9.31 (1H, s, D₂O exch., NH). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 53.8 (CH₂), 55.0 (OCH₃), 94.3 (C-4), 114.2 (C-3″, C-5″), 118.3 (C-2′, C-6′), 121.0 (C-4′), 123.8 (C-3a), 126.9 (C-1″), 128.7 (C-3′, C-5′), 129.7 (C-2″, C-6″), 133.6 (C-7a), 134.7 (C-7), 141.4 (C-1′), 145.6 (C-3), 152.9 (C-5), 159.2 (C-4″). Anal. Calcd for C₂₀H₁₇N₅O₃: C, 63.99; H, 4.56; N, 18.66. Found: C, 64.06; H, 4.67; N, 18.58.

N⁵-Cyclohexyl-1-methyl-1H-pyrazolo[3,4-c]pyridine-3,5-diamine (11a)

A solution of nitro derivative 10a (400 mg, 1.45 mmol) in absolute ethanol (40 mL) was hydrogenated in the presence of 10% Pd/C (80 mg) under a pressure of 55 psi at room temperature (r.t.) for 3h. The solution was filtered through a celite pad to remove the catalyst and the filtrate was evaporated to dryness. The residue was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (99 : 1, v/v) as the eluent, to provide pure 11a (280 mg, 79%) as a yellow solid. mp: 179–180°C (EtOAc). ¹H-NMR (400 MHz, CDCl₃) δ: 1.11–1.24 (3H, m, cyclohexyl H), 1.32 (2H, m, cyclohexyl H), 1.60 (1H, m, cyclohexyl H), 1.72 (2H, m, cyclohexyl H), 2.02 (2H, m, cyclohexyl H), 3.29 (1H, m, cyclohexyl H-1′), 3.80 (3H, s, CH₃), 4.18 (3H, br s, D₂O exch., NH₂, NH), 6.33 (1H, d, H-4, J=1.0 Hz), 8.28 (1H, d, H-7, J=1.0 Hz). ¹³C-NMR (50 MHz, CDCl₃) δ: 24.9 (cyclohexyl C-3′, C-5′), 25.8 (cyclohexyl C-4′), 33.1 (cyclohexyl C-2′, C-6′), 35.3 (CH₃), 51.7 (cyclohexyl C-1′), 91.5 (C-4), 122.5 (C-3a), 130.3 (C-7), 134.3 (C-7a), 145.4 (C-3), 150.9 (C-5). Anal. Calcd for C₁₃H₁₉N₅: C, 63.65; H, 7.81; N, 28.55. Found: C, 63.97; H, 8.08; N, 28.29.

1-Methyl-N⁵-phenyl-1H-pyrazolo[3,4-c]pyridine-3,5-diamine (11b)

This compound was prepared following an analogous procedure to that described for the synthesis of 11a, starting from the nitro derivative 10b. The product was purified by column chromatography (silica gel) using a mixture of cyclohexane–ethyl acetate (from 5 : 5 up to 2 : 8, v/v) as the eluent, to provide pure 11b as a green solid, in 84% yield. mp: 178–179°C (EtOAc). ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.75 (3H, s, CH₃), 5.61 (2H, br s, D₂O exch., NH₂), 6.78 (1H, t, H-4′, J=7.2 Hz), 7.21 (3H, m, H-3′, H-5′, H-4), 7.37 (2H, dd, H-2′, H-6′, J=8.0 Hz, J=0.9 Hz), 8.56 (2H, m, H-7, NH-aniline). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 35.1 (CH₃), 97.9 (C-4), 116.2 (C-2′, C-6′), 118.8 (C-4′), 121.1 (C-3a), 128.6 (C-3′, C-5′), 130.4 (C-7), 134.8 (C-7a), 143.5 (C-1′), 146.4 (C-5), 147.3 (C-3). Anal. Calcd for C₁₃H₁₃N₅: C, 65.26; H, 5.48; N, 29.27. Found: C, 65.03; H, 5.74; N, 29.49.

N⁵-Cyclohexyl-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-c]pyridine-3,5-diamine (11c)

This compound was prepared following an analogous procedure to that described for the synthesis of 11a, starting from the nitro derivative 10c. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–ethyl acetate (8 : 2, v/v) as the eluent, providing 11c as a yellow solid, in 85% yield. mp: 142–143°C (Et₂O). ¹H-NMR (400 MHz, CDCl₃) δ: 1.20 (3H, m, cyclohexyl H), 1.33 (2H, m, cyclohexyl H), 1.60 (1H, m, cyclohexyl H), 1.72 (2H, m, cyclohexyl H), 2.02 (2H, m, cyclohexyl H), 3.29 (1H, m, cyclohexyl H-1′), 3.72 (3H, s, OCH₃), 4.14 (2H, br s, D₂O exch., NH₂), 5.21 (2H, s, CH₂), 6.32 (1H, d, H-4, J=1.0 Hz), 6.78 (2H, d, H-3″, H-5″, J=8.7 Hz), 7.13 (2H, d, H-2″, H-6″, J=8.7 Hz), 8.20 (1H, d, H-7, J=1.0 Hz). ¹³C-NMR (50 MHz, CDCl₃) δ: 25.0 (cyclohexyl C-3′, C-5′), 25.9 (cyclohexyl C-4′), 33.2 (cyclohexyl C-2′, C-6′), 51.6 (cyclohexyl C-1′), 52.6 (CH₂), 55.3 (OCH₃), 91.2 (C-4), 114.1 (C-3″, C-5″), 123.0 (C-3a), 128.8 (C-2″, C-6″), 129.2 (C-1″), 131.4 (C-7), 133.9 (C-7a), 145.8 (C-3), 151.4 (C-5), 159.2 (C-4″). Anal. Calcd for C₂₀H₂₅N₅O: C, 68.35; H, 7.17; N, 19.93. Found: C, 68.08; H, 7.04; N, 20.22.

1-(4-Methoxybenzyl)-N⁵-phenyl-1H-pyrazolo[3,4-c]pyridine-3,5-diamine (11d)

Ten percent Pd/C (5 mg) and triethylsilane (0.34 mL, 2.13 mmol) were added into a solution of the nitro derivative 10d (80 mg, 0.21 mmol) in anhydrous methanol (5 mL), under argon, and the resulting solution was stirred at r.t. for 12h. The solution was filtered through a celite pad to remove the catalyst and the filtrate was evaporated to dryness. The residue was purified by column chromatography (silica gel) using a mixture of cyclohexane–ethyl acetate (8 : 2, v/v) as the eluent, to provide pure 11d (60 mg, 82%) as a brown solid. mp: 148°C (Et₂O). ¹H-NMR (400 MHz, CDCl₃) δ: 3.78 (3H, s, OCH₃), 4.64 (2H, br s, D₂O exch., NH₂), 5.27 (2H, s, CH₂), 6.84 (2H, d, H-3″, H-5″, J=8.6 Hz), 7.06 (2H, m, H-4′, H-4), 7.18 (2H, d, H-2″, H-6″, J=8.6 Hz), 7.23 (2H, d, H-2′, H-6′, J=7.6 Hz), 7.32 (2H, t, H-3′, H-5′, J=7.6 Hz), 7.58 (1H, br s, D₂O exch., NH), 8.20 (1H, s, H-7). ¹³C-NMR (50 MHz, CDCl₃) δ: 53.1 (CH₂), 55.3 (OCH₃), 96.7 (C-4), 114.3 (C-3″, C-5″), 120.6 (C-2′, C-6′), 123.5 (C-4′), 124.2 (C-3a), 127.0 (C-7), 127.9 (C-1″), 128.9 (C-2″, C-6″), 129.6 (C-3′, C-5′), 133.2 (C-7a), 140.1 (C-1′), 146.2 (C-3), 146.5 (C-5), 159.6 (C-4″). Anal. Calcd for C₂₀H₁₉N₅O: C, 69.55; H, 5.54; N, 20.28. Found: C, 69.29; H, 5.37; N, 20.46.

General Procedure for the Synthesis of Derivatives 12a–d

Chloroacetyl chloride (96 µL, 1.2 mmol) and triethylamine (153 µL, 1.1 mmol) were added into a solution of the amines 11a–d (1 mmol) in anhydrous tetrahydrofuran (THF) (15 mL), under argon, at −40°C for amines 11a, c or at 0°C for amines 11b, d. The resulting solution was allowed to reach r.t. and stirring was continued for one more hour. The solvents were then vacuum-evaporated and the residue was used imediately to the next step, without any further purification.

General Procedure for the Synthesis of Derivatives 13a–l

Dimethylamine (5.6 M solution in ethanol, 0.89 mL, 5 mmol) or 1-methylpiperazine (0.22 mL, 2 mmol) or aniline (0.36 mL, 4 mmol) was added into a solution of the intermediate chlorides 12a–d (1 mmol) in absolute ethanol (10 mL), under argon, and the resulting solution was heated at 80°C for 4 h. Upon completion of the reaction solvents were evaporated and the residue was purified by column chromatography to provide pure amides 13a–l.

N-(5-(Cyclohexylamino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(dimethylamino)acetamide (13a)

This compound was prepared according to the general procedure described above, upon reaction of the chloride 12a with dimethylamine (5.6 M solution in ethanol). The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (100 : 1, v/v) as the eluent to provide pure 13a as a green oil. Yield: 52%. ¹H-NMR (400 MHz, CDCl₃) δ: 1.22 (3H, m, cyclohexyl H), 1.40 (2H, m, cyclohexyl H), 1.61 (1H, m, cyclohexyl H), 1.73 (2H, m, cyclohexyl H), 2.05 (2H, m, cyclohexyl H), 2.42 (6H, s, N(CH₃)₂), 3.17 (2H, s, CH₂), 3.44 (1H, m, cyclohexyl H-1′), 3.94 (3H, s, CH₃–pyrazole), 6.83 (1H, s, H-4), 8.39 (1H, s, H-7), 9.55 (1H, s, D₂O exch., NHCO). ¹³C-NMR (50 MHz, CDCl₃) δ: 24.8 (cyclohexyl C-3′, C-5′), 25.9 (cyclohexyl C-4′), 33.2 (cyclohexyl C-2′, C-6′), 35.7 (CH₃–pyrazole), 46.0 (N(CH₃)₂), 51.2 (cyclohexyl C-1′), 63.0 (CH₂), 93.8 (C-4), 123.9 (C-3a), 131.3 (C-7), 134.2 (C-7a), 136.9 (C-3), 152.0 (C-5), 168.6 (CO). HR-MS (electrospray ionization (ESI)) m/z: Calcd for C₁₇H₂₇N₆O: [M1+H]⁺=331.2241. Found 331.2247. Anal. Calcd for C₁₇H₂₆N₆O: C, 61.79; H, 7.93; N, 25.43. Found: C, 61.69; H, 7.76; N, 25.55.

N-(5-(Cyclohexylamino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(4-methylpiperazin-1-yl)acetamide (13b)

This compound was prepared according to the general procedure described above, upon reaction of the chloride 12a with 1-methylpiperazine. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (94 : 6, v/v) as the eluent to provide pure 13b as a yellow solid. Yield: 38%, mp: 154°C (dec.) (Et₂O). ¹H-NMR (400 MHz, CDCl₃) δ: 1.23 (3H, m, cyclohexyl H), 1.40 (2H, m, cyclohexyl H), 1.62 (1H, m, cyclohexyl H), 1.74 (2H, m, cyclohexyl H), 2.06 (2H, m, cyclohexyl H), 2.39 (3H, s, CH₃–piperazine), 2.63 (4H, br s, piperazine H-3″, H-5″), 2.77 (4H, br s, piperazine H-2″, H-6″), 3.24 (2H, s, CH₂), 3.45 (1H, m, cyclohexyl H-1′), 3.96 (3H, s, CH₃–pyrazole), 6.78 (1H, d, H-4, J=1.0 Hz), 8.41 (1H, d, H-7, J=1.0 Hz), 9.40 (1H, s, D₂O exch., NHCO). ¹³C-NMR (50 MHz, CDCl₃) δ: 24.9 (cyclohexyl C-3′, C-5′), 26.0 (cyclohexyl C-4′), 33.3 (cyclohexyl C-2′, C-6′), 35.8 (CH₃–pyrazole), 45.7 (CH₃–piperazine), 51.3 (cyclohexyl C-1′), 53.1 (piperazine C-2″, C-6″), 55.0 (piperazine C-3″, C-5″), 61.5 (CH₂), 93.6 (C-4), 124.0 (C-3a), 131.6 (C-7), 134.3 (C-7a), 136.8 (C-3), 152.1 (C-5), 168.2 (CO). HR-MS (ESI) m/z: Calcd for C₂₀H₃₂N₇O: [M1+H]⁺=386.2663. Found 386.2665. Anal. Calcd for C₂₀H₃₁N₇O: C, 62.31; H, 8.11; N, 25.43. Found: C, 62.56; H, 8.22; N, 25.29.

N-(5-(Cyclohexylamino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(phenylamino)acetamide (13c)

This compound was prepared according to the general procedure described above, upon reaction of the chloride 12a with aniline. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (100 : 1, v/v) as the eluent to provide pure 13c as a yellow solid. Yield: 35%, mp: 175–176°C (Et₂O). ¹H-NMR (400 MHz, CDCl₃) δ: 1.20–1.31 (3H, m, cyclohexyl H), 1.42 (2H, m, cyclohexyl H), 1.66 (1H, m, cyclohexyl H), 1.78 (2H, m, cyclohexyl H), 2.07 (2H, m, cyclohexyl H), 3.44 (1H, m, cyclohexyl H-1′), 3.91 (3H, s, CH₃), 4.00 (2H, s, CH₂), 4.57 (1H, br s, D₂O exch., NH-cyclohexylamine), 6.71 (2H, d, H-2″, H-6″, J=7.9 Hz), 6.74 (1H, s, H-4), 6.85 (1H, t, H-4″, J=7.3 Hz), 7.24 (2H, t, H-3″, H-5″, J=7.5 Hz), 8.38 (1H, s, H-7), 9.10 (1H, br s, D₂O exch., NHCO). ¹³C-NMR (50 MHz, CDCl₃) δ: 24.8 (cyclohexyl C-3′, C-5′), 25.9 (cyclohexyl C-4′), 33.1 (cyclohexyl C-2′, C-6′), 35.7 (CH₃), 49.1 (CH₂), 51.2 (cyclohexyl C-1′), 93.2 (C-4), 113.4 (C-2″, C-6″), 119.5 (C-4″), 124.4 (C-3a), 129.5 (C-3″, C-5″), 131.1 (C-7), 134.0 (C-7a), 136.4 (C-3), 147.0 (C-1″), 151.9 (C-5), 169.2 (CO). HR-MS (ESI) m/z: Calcd for C₂₁H₂₇N₆O: [M1+H]⁺=379.2241. Found 379.2244. Anal. Calcd for C₂₁H₂₆N₆O: C, 66.64; H, 6.92; N, 22.20. Found: C, 66.48; H, 6.69; N, 22.43.

2-(Dimethylamino)-N-(1-methyl-5-(phenylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)acetamide (13d)

This compound was prepared according to the general procedure described above, upon reaction of the chloride 12b with dimethylamine (5.6 M solution in ethanol). The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (100 : 2, v/v) as the eluent to provide pure 13d as a green solid. Yield: 86%, mp: 166–167°C (EtOH). ¹H-NMR (400 MHz, CDCl₃) δ: 2.41 (6H, s, N(CH₃)₂), 3.15 (2H, s, CH₂), 4.00 (3H, s, CH₃–pyrazole), 6.64 (1H, s, D₂O exch., NH–aniline), 6.95 (1H, t, H-4′, J=7.2 Hz), 7.30 (4H, m, H-2′, H-3′, H-5′, H-6′), 7.57 (1H, d, H-4, J=1.2 Hz), 8.54 (1H, d, H-7, J=1.2 Hz), 9.59 (1H, s, D₂O exch., NHCO). ¹³C-NMR (50 MHz, CDCl₃) δ: 35.7 (CH₃–pyrazole), 46.0 (N(CH₃)₂), 63.0 (CH₂), 98.7 (C-4), 117.8 (C-2′, C-6′), 121.1 (C-4′), 123.2 (C-3a), 129.2 (C-3′, C-5′), 131.5 (C-7), 135.0 (C-7a), 137.5 (C-3), 142.1 (C-1′), 147.9 (C-5), 168.8 (CO). HR-MS (ESI) m/z: Calcd for C₁₇H₂₁N₆O: [M1+H]⁺=325.1771. Found 325.1778. Anal. Calcd for C₁₇H₂₀N₆O: C, 62.95; H, 6.21; N, 25.91. Found: C, 63.04; H, 6.26; N, 25.78.

N-(1-Methyl-5-(phenylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(4-methylpiperazin-1-yl)acetamide (13e)

This compound was prepared according to the general procedure described above, upon reaction of the chloride 12b with 1-methylpiperazine. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (9 : 1, v/v) as the eluent to provide pure 13e as a yellow solid. Yield: 93%, mp: 196–197°C (EtOH). ¹H-NMR (400 MHz, DMSO-d₆) δ: 2.18 (3H, s, CH₃–piperazine), 2.38 (4H, br s, piperazine H-3″, H-5″), 2.57 (4H, br s, piperazine H-2″, H-6″), 3.20 (2H, s, CH₂), 4.01 (3H, s, CH₃–pyrazole), 6.80 (1H, t, H-4′, J=7.2 Hz), 7.21 (3H, m, H-3′, H-5′, H-4), 7.45 (2H, d, H-2′, H-6′, J=8.0 Hz), 8.74 (1H, s, D₂O exch., NH–aniline), 8.80 (1H, d, H-7, J=1.0 Hz), 10.14 (1H, br s, D₂O exch., NHCO). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 35.5 (CH₃–pyrazole), 45.6 (CH₃–piperazine), 52.6 (piperazine C-2″, C-6″), 54.5 (piperazine C-3″, C-5″), 60.8 (CH₂), 98.4 (C-4), 116.5 (C-2′, C-6′), 119.2 (C-4′), 123.2 (C-3a), 128.6 (C-3′, C-5′), 131.8 (C-7), 134.2 (C-7a), 137.2 (C-3), 143.1 (C-1′), 147.8 (C-5), 168.2 (CO). HR-MS (ESI) m/z: Calcd for C₂₀H₂₆N₇O: [M1+H]⁺=380.2193. Found 380.2200. Anal. Calcd for C₂₀H₂₅N₇O: C, 63.30; H, 6.64; N, 25.84. Found: C, 63.41; H, 6.79; N, 25.73.

N-(1-Methyl-5-(phenylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(phenylamino)acetamide (13f)

This compound was prepared according to the general procedure described above, upon reaction of the chloride 12b with aniline. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–ethyl acetate (1 : 1, v/v) as the eluent to provide pure 13f as a yellow solid. Yield: 70%, mp: 180–181°C (EtOH). ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.95 (2H, d, CH₂, J=6.0 Hz), 4.01 (3H, s, CH₃), 6.04 (1H, t, CH₂–NH–, J=6.0 Hz), 6.60 (3H, m, H-2″, H-4″, H-6″), 6.80 (1H, t, H-4′, J=7.2 Hz), 7.11 (2H, t, H-3″, H-5″, J=7.7 Hz), 7.20 (3H, m, H-3′, H-5′, H-4), 7.42 (2H, d, H-2′, H-6′, J=7.8 Hz), 8.72 (1H, s, D₂O exch., NH–aniline), 8.79 (1H, d, H-7, J=0.8 Hz), 10.50 (1H, br s, D₂O exch., NHCO). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 36.1 (CH₃), 47.0 (CH₂), 98.9 (C-4), 112.7 (C-2″, C-6″), 116.9 (C-4″), 117.0 (C-2′, C-6′), 119.7 (C-4′), 123.6 (C-3a), 129.1 (C-3′, C-5′), 129.4 (C-3″, C-5″), 132.4 (C-7), 134.7 (C-7a), 137.9 (C-3), 143.7 (C-1′), 148.3 (C-5), 148.7 (C-1″), 169.9 (CO). HR-MS (ESI) m/z: Calcd for C₂₁H₂₁N₆O: [M1+H]⁺=373.1771. Found 373.1772. Anal. Calcd for C₂₁H₂₀N₆O: C, 67.73; H, 5.41; N, 22.57. Found: C, 67.94; H, 5.56; N, 22.28.

N-(5-(Cyclohexylamino)-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(dimethylamino)acetamide (13g)

This compound was prepared according to the general procedure described above, upon reaction of the chloride 12c with dimethylamine (5.6 M solution in ethanol). The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (100 : 1, v/v) as the eluent to provide pure 13g as a yellow oil. Yield: 57%. ¹H-NMR (400 MHz, CDCl₃) δ: 1.18 (3H, m, cyclohexyl H), 1.36 (2H, m, cyclohexyl H), 1.57 (1H, m, cyclohexyl H), 1.69 (2H, m, cyclohexyl H), 2.01 (2H, m, cyclohexyl H), 2.36 (6H, s, N(CH₃)₂), 3.11 (2H, s, NHCOCH₂), 3.42 (1H, m, cyclohexyl H-1″), 3.70 (3H, s, OCH₃), 4.30 (1H, br s, D₂O exchang., NH–cyclohexylamine), 5.32 (2H, s, CH₂–pyrazole), 6.77 (2H, d, H-3′, H-5′, J=8.6 Hz), 6.84 (1H, s, H-4), 7.11 (2H, d, H-2′, H-6′, J=8.6 Hz), 8.28 (1H, s, H-7), 9.55 (1H, s, D₂O exchang., NHCO). ¹³C-NMR (50 MHz, CDCl₃) δ: 24.6 (cyclohexyl C-3″, C-5″), 25.7 (cyclohexyl C-4″), 33.0 (cyclohexyl C-2″, C-6″), 45.9 (N(CH₃)₂), 50.9 (cyclohexyl C-1″), 52.7 (CH₂–pyrazole), 55.0 (OCH₃), 62.9 (NHCOCH₂), 93.9 (C-4), 114.0 (C-3′, C-5′), 124.2 (C-3a), 128.1 (C-1′), 128.5 (C-2′, C-6′), 131.6 (C-7), 133.5 (C-7a), 137.3 (C-3), 151.8 (C-5), 159.2 (C-4′), 168.5 (CO). HR-MS (ESI) m/z: Calcd for C₂₄H₃₃N₆O₂: [M1+H]⁺=437.2660. Found 437.2666. Anal. Calcd for C₂₄H₃₂N₆O₂: C, 66.03; H, 7.39; N, 19.25. Found: C, 66.21; H, 7.52; N, 19.01.

N-(5-(Cyclohexylamino)-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(4-methylpiperazin-1-yl)acetamide (13h)

This compound was prepared according to the general procedure described above, upon reaction of the chloride 12c with 1-methylpiperazine. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (96 : 4, v/v) as the eluent to provide pure 13h as a yellow solid. Yield: 98%, mp: 157–158°C (CHCl₃/Et₂O). ¹H-NMR (400 MHz, CDCl₃) δ: 1.21 (3H, m, cyclohexyl H), 1.40 (2H, m, cyclohexyl H), 1.62 (1H, m, cyclohexyl H), 1.74 (2H, m, cyclohexyl H), 2.04 (2H, m, cyclohexyl H), 2.47 (3H, s, CH₃–piperazine), 2.74 (4H, br s, piperazine H-3″′, H-5‴), 2.84 (4H, br s, piperazine H-2″′, H-6‴), 3.26 (2H, s, NHCOCH₂), 3.44 (1H, m, cyclohexyl H-1″), 3.77 (3H, s, OCH₃), 4.29 (1H, br s, D₂O exchang., NH–cyclohexylamine), 5.37 (2H, s, CH₂–pyrazole), 6.80 (1H, d, H-4, J=1.0 Hz), 6.83 (2H, d, H-3′, H-5′, J=8.7 Hz), 7.16 (2H, d, H-2′, H-6′, J=8.7 Hz), 8.31 (1H, d, H-7, J=1.0 Hz), 9.34 (1H, s, D₂O exchang., NHCO). ¹³C-NMR (50 MHz, CDCl₃) δ: 24.9 (cyclohexyl C-3″, C-5″), 26.0 (cyclohexyl C-4″), 33.3 (cyclohexyl C-2″, C-6″), 45.4 (CH₃–piperazine), 51.2 (cyclohexyl C-1″), 52.7 (piperazine C-2″′, C-6‴), 53.1 (CH₂–pyrazole), 54.8 (piperazine C-3″′, C-5‴), 55.4 (OCH₃), 61.5 (NHCOCH₂), 94.0 (C-4), 114.4 (C-3′, C-5′), 124.6 (C-3a), 128.4 (C-1′), 128.9 (C-2″, C-6″), 132.1 (C-7), 133.8 (C-7a), 137.3 (C-3), 152.2 (C-5), 159.6 (C-4″), 168.0 (CO). HR-MS (ESI) m/z: Calcd for C₂₇H₃₈N₇O₂: [M1+H]⁺=492.3081. Found 492.3085. Anal. Calcd for C₂₇H₃₇N₇O₂: C, 65.96; H, 7.59; N, 19.94. Found: C, 65.78; H, 7.51; N, 20.20.

N-(5-(Cyclohexylamino)-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(phenylamino)acetamide (13i)

This compound was prepared according to the general procedure described above, upon reaction of the chloride 12c with aniline. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (100 : 1, v/v) as the eluent to provide pure 13i as a yellow solid. Yield: 46%, mp: 152°C (CHCl₃/Et₂O). ¹H-NMR (400 MHz, CDCl₃) δ: 1.24 (3H, m, cyclohexyl H), 1.40 (2H, m, cyclohexyl H), 1.64 (1H, m, cyclohexyl H), 1.75 (2H, m, cyclohexyl H), 2.05 (2H, m, cyclohexyl H), 3.44 (1H, m, cyclohexyl H-1″), 3.74 (3H, s, OCH₃), 3.97 (2H, d, COCH₂NH, J=5.2 Hz), 4.32 (1H, br s, D₂O exchang., NH–cyclohexylamine), 4.45 (1H, t, D₂O exchang., COCH₂NH, J=5.2 Hz), 5.31 (2H, s, CH₂–pyrazole), 6.70 (2H, d, H-2″′, H-6″′, J=7.7 Hz), 6.76 (1H, s, H-4), 6.78 (2H, d, H-3′, H-5′, J=8.5 Hz), 6.84 (1H, t, H-4″′, J=7.2 Hz), 7.11 (2H, d, H-2′, H-6′, J=8.5 Hz), 7.23 (2H, t, H-3″′, H-5″′, J=7.5 Hz), 8.30 (1H, s, H-7), 9.05 (1H, s, D₂O exchang., NHCO). ¹³C-NMR (50 MHz, CDCl₃) δ: 24.8 (cyclohexyl C-3″, C-5″), 25.9 (cyclohexyl C-4″), 33.0 (cyclohexyl C-2″, C-6″), 49.3 (NHCOCH₂), 51.2 (cyclohexyl C-1″), 53.2 (CH₂–pyrazole), 55.3 (OCH₃), 94.1 (C-4), 113.5 (C-2″′, C-6‴), 114.3 (C-3′, C-5′), 119.7 (C-4‴), 125.0 (C-3a), 127.9 (C-1′), 128.8 (C-2′, C-6′), 129.6 (C-3″′, C-5‴), 130.9 (C-7), 133.3 (C-7a), 136.9 (C-3), 146.9 (C-1‴), 151.6 (C-5), 159.5 (C-4′), 169.0 (CO). HR-MS (ESI) m/z: Calcd for C₂₈H₃₃N₆O₂: [M1+H]⁺=485.2660. Found 485.2663. Anal. Calcd for C₂₈H₃₂N₆O₂: C, 69.40; H, 6.66; N, 17.34. Found: C, 69.63; H, 6.80; N, 17.12.

2-(Dimethylamino)-N-(1-(4-methoxybenzyl)-5-phenylamino-1H-pyrazolo[3,4-c]pyridin-3-yl)acetamide (13j)

This compound was prepared according to the general procedure described above, upon reaction of the chloride 12d with dimethylamine (5.6 M solution in ethanol). The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–ethyl acetate (6 : 4, v/v) as the eluent to provide pure 13j as a pale green solid. Yield: 70%, mp: 143°C (Et₂O). ¹H-NMR (400 MHz, CDCl₃) δ: 2.36 (6H, s, N(CH₃)₂), 3.12 (2H, s, NHCOCH₂), 3.72 (3H, s, OCH₃), 5.36 (2H, s, CH₂–pyrazole), 6.82 (2H, d, H-3″, H-5″, J=8.7 Hz), 6.90 (2H, m, H-4′, NH–aniline), 7.17 (2H, d, H-2″, H-6″, J=8.7 Hz), 7.26 (4H, m, H-2′, H-3′, H-5′, H-6′), 7.59 (1H, d, H-4, J=1.0 Hz), 8.45 (1H, d, H-7, J=1.0 Hz), 9.69 (1H, br s, D₂O exch., NHCO). ¹³C-NMR (50 MHz, CDCl₃) δ: 46.0 (N(CH₃)₂), 53.0 (CH₂–pyrazole), 55.3 (OCH₃), 63.0 (NHCOCH₂), 99.1 (C-4), 114.3 (C-3″, C-5″), 117.9 (C-2′, C-6′), 121.1 (C-4′), 123.8 (C-3a), 128.1 (C-1″), 128.9 (C-2″, C-6″), 129.2 (C-3′, C-5′), 131.9 (C-7), 134.5 (C-7a), 138.1 (C-3), 142.2 (C-1′), 148.1 (C-5), 159.5 (C-4″), 168.9 (CO). HR-MS (ESI) m/z: Calcd for C₂₄H₂₇N₆O₂: [M1+H]⁺=431.2190. Found 431.2191. Anal. Calcd for C₂₄H₂₆N₆O₂: C, 66.96; H, 6.09; N, 19.52. Found: C, 67.11; H, 6.24; N, 19.39.

N-(1-(4-Methoxybenzyl)-5-(phenylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(4-methylpiperazin-1-yl)acetamide (13k)

This compound was prepared according to the general procedure described above, upon reaction of the chloride 12d with 1-methylpiperazine. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (98 : 2, v/v) as the eluent to provide pure 13k as a pale green solid. Yield: 70%, mp: 134°C (CH₂Cl₂/Et₂O). ¹H-NMR (400 MHz, CDCl₃) δ: 2.31 (3H, s, CH₃–piperazine), 2.52 (4H, br s, piperazine H-3″′, H-5‴), 2.68 (4H, br s, piperazine H-2″′, H-6‴), 3.18 (2H, s, NHCOCH₂), 3.74 (3H, s, OCH₃), 5.40 (2H, s, CH₂–pyrazole), 6.74 (1H, br s, NH), 6.82 (2H, d, H-3″, H-5″, J=8.7 Hz), 6.92 (1H, m, H-4′), 7.17 (2H, d, H-2″, H-6″, J=8.7 Hz), 7.26 (4H, m, H-2′, H-3′, H-5′, H-6′), 7.51 (1H, d, H-4, J=1.0 Hz), 8.42 (1H, d, H-7, J=1.0 Hz), 9.51 (1H, br s, D₂O exch., NHCO). ¹³C-NMR (50 MHz, CDCl₃) δ: 45.8 (CH₃–piperazine), 53.1 (CH₂–pyrazole), 53.3 (piperazine C-2″′, C-6‴), 54.9 (piperazine C-3″′, C-5‴), 55.3 (OCH₃), 61.4 (NHCOCH₂), 98.7 (C-4), 114.3 (C-3″, C-5″), 118.0 (C-2′, C-6′), 121.2 (C-4′), 123.9 (C-3a), 128.0 (C-1″), 128.8 (C-2″, C-6″), 129.2 (C-3′, C-5′), 132.0 (C-7), 134.5 (C-7a), 137.8 (C-3), 142.1 (C-1′), 148.1 (C-5), 159.5 (C-4″), 168.4 (CO). HR-MS (ESI) m/z: Calcd for C₂₇H₃₂N₇O₂: [M1+H]⁺=486.2612. Found 486.2619. Anal. Calcd for C₂₇H₃₁N₇O₂: C, 66.78; H, 6.43; N, 20.19. Found: C, 66.94; H, 6.33; N, 20.07.

N-(1-(4-Methoxybenzyl)-5-(phenylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(phenylamino)acetamide (13l)

This compound was prepared according to the general procedure described above, upon reaction of the chloride 12d with aniline. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (100 : 1, v/v) as the eluent to provide pure 13l as a beige solid. Yield: 68%, mp: 208–209°C (CHCl₃/Et₂O). ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.70 (3H, s, OCH₃), 3.93 (2H, br s, NHCOCH₂), 5.50 (2H, s, CH₂–pyrazole), 6.00 (1H, br s, CH₂NH), 6.58 (1H, t, H-4″′, J=7.2 Hz), 6.61 (2H, d, H-2″′, H-6″′, J=7.8 Hz), 6.79 (1H, t, H-4′, J=7.2 Hz), 6.88 (2H, d, H-3″, H-5″, J=8.5 Hz), 7.10 (2H, t, H-3″′, H-5″′, J=7.5 Hz), 7.19 (3H, m, H-3′, H-5′, H-4), 7.27 (2H, d, H-2″, H-6″, J=8.5 Hz), 7.41 (2H, d, H-2′, H-6′, J=8.2 Hz), 8.73 (1H, s, D₂O exch., NH–aniline), 8.86 (1H, s, H-7), 10.57 (1H, s, D₂O exch., NHCO). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 46.4 (NHCOCH₂), 51.6 (CH₂–pyrazole), 55.0 (OCH₃), 98.7 (C-4), 112.2 (C-2″′, C-6‴), 113.9 (C-3″, C-5″), 116.3 (C-4‴), 116.6 (C-2′, C-6′), 119.2 (C-4′), 123.5 (C-3a), 128.6 (C-3′, C-5′), 128.9 (C-3″′, C-5‴), 129.0 (C-1″), 129.2 (C-2″, C-6″), 131.9 (C-7), 133.6 (C-7a), 138.0 (C-3), 143.0 (C-1′), 147.9 (C-5), 148.1 (C-1‴), 158.8 (C-4″), 169.4 (CO). HR-MS (ESI) m/z: Calcd for C₂₈H₂₇N₆O₂: [M1+H]⁺=479.2190. Found 479.2194. Anal. Calcd for C₂₈H₂₆N₆O₂: C, 70.28; H, 5.48; N, 17.56. Found: C, 70.03; H, 5.39; N, 17.66.

General Procedure for the Synthesis of Derivatives 14a–f

A solution of the compounds 13g–l (0.2 mmol) in trifluoroacetic acid (5 mL) was heated at 70°C for 96 h. Then the solvent was vacuum-evaporated and the residue was neutralized with sodium bicarbonate and extracted with ethyl acetate (3×50 mL). The combined organic extracts were dried (Na₂SO₄), concentrated under vacuum and the residue was purified by column chromatography to provide pure target compounds 14a–f.

N-(5-(Cyclohexylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(dimethylamino)acetamide (14a)

This compound was prepared according to the general procedure described above, starting from 13g. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (95 : 5, v/v) as the eluent to provide pure 14a as a green solid. Yield: 80%, mp: 132–133°C (Et₂O/n-pentane). ¹H-NMR (400 MHz, CDCl₃) δ: 1.18 (3H, m, cyclohexyl H), 1.32 (2H, m, cyclohexyl H), 1.57 (1H, m, cyclohexyl H), 1.68 (2H, m, cyclohexyl H), 2.01 (2H, m, cyclohexyl H), 2.38 (6H, s, N(CH₃)₂), 3.17 (2H, s, CH₂), 3.40 (1H, m, cyclohexyl H-1′), 4.23 (1H, br s, D₂O exchang., NH–cyclohexylamine), 6.80 (1H, s, H-4), 8.37 (1H, s, H-7), 9.62 (1H, br s, D₂O exchang., NHCO), 11.62 (1H, br s, D₂O exchang., NH–pyrazole). ¹³C-NMR (50 MHz, CDCl₃) δ: 24.7 (cyclohexyl C-3′, C-5′), 25.8 (cyclohexyl C-4′), 33.0 (cyclohexyl C-2′, C-6′), 46.0 (N(CH₃)₂), 51.2 (cyclohexyl C-1′), 63.0 (CH₂), 93.3 (C-4), 123.3 (C-3a), 132.6 (C-7), 134.2 (C-7a), 138.4 (C-3), 151.8 (C-5), 169.2 (CO). HR-MS (ESI) m/z: Calcd for C₁₆H₂₅N₆O: [M1+H]⁺=317.2084. Found 317.2088. Anal. Calcd for C₁₆H₂₄N₆O: C, 60.74; H, 7.65; N, 26.56. Found: C, 61.01; H, 7.80; N, 26.33.

N-(5-(Cyclohexylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(4-methylpiperazin-1-yl)acetamide (14b)

This compound was prepared according to the general procedure described above, starting from 13h. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (10 : 1, v/v) as the eluent to provide pure 14b as a pale yellow solid. Yield: 88%, mp: 145°C (Et₂O). ¹H-NMR (400 MHz, CDCl₃) δ: 1.23 (3H, m, cyclohexyl H), 1.40 (2H, m, cyclohexyl H), 1.63 (1H, m, cyclohexyl H), 1.74 (2H, m, cyclohexyl H), 2.06 (2H, m, cyclohexyl H), 2.33 (3H, s, CH₃), 2.54 (4H, br s, piperazine H-3″, H-5″), 2.72 (4H, br s, piperazine H-2″, H-6″), 3.24 (2H, s, CH₂), 3.44 (1H, m, cyclohexyl H-1′), 4.33 (1H, br s, D₂O exchang., NH–cyclohexylamine), 6.80 (1H, s, H-4), 8.44 (1H, s, H-7), 9.55 (1H, s, D₂O exchang., NHCO), 10.52 (1H, br s, D₂O exchang., NH–pyrazole). ¹³C-NMR (50 MHz, CDCl₃) δ: 24.8 (cyclohexyl C-3′, C-5′), 25.9 (cyclohexyl C-4′), 33.2 (cyclohexyl C-2′, C-6′), 45.9 (CH₃), 51.2 (cyclohexyl C-1′), 53.5 (piperazine C-2″, C-6″), 55.1 (piperazine C-3″, C-5″), 61.5 (CH₂), 93.3 (C-4), 123.6 (C-3a), 132.4 (C-7), 134.2 (C-7a), 139.0 (C-3), 152.2 (C-5), 168.5 (CO). HR-MS (ESI) m/z: Calcd for C₁₉H₃₀N₇O: [M1+H]⁺=372.2506. Found 372.2511. Anal. Calcd for C₁₉H₂₉N₇O: C, 61.43; H, 7.87; N, 26.39. Found: C, 61.19; H, 7.76; N, 26.48.

N-(5-(Cyclohexylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(phenylamino)acetamide (14c)

This compound was prepared according to the general procedure described above, starting from 13i. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (96 : 4, v/v) as the eluent to provide pure 14c as a yellow solid. Yield: 74%, mp: 166°C (CHCl₃). ¹H-NMR (400 MHz, CDCl₃) δ: 1.23 (3H, m, cyclohexyl H), 1.41 (2H, m, cyclohexyl H), 1.64 (1H, m, cyclohexyl H), 1.75 (2H, m, cyclohexyl H), 2.05 (2H, m, cyclohexyl H), 3.41 (1H, m, cyclohexyl H-1′), 4.01 (2H, s, CH₂), 4.49 (1H, s, D₂O exchang., CH₂NH), 6.69 (1H, s, H-4), 6.72 (2H, d, H-2″, H-6″, J=8.0 Hz), 6.85 (1H, t, H-4″, J=7.2 Hz), 7.24 (2H, t, H-3″, H-5″, J=7.7 Hz), 8.42 (1H, s, H-7), 9.07 (1H, s, D₂O exchang., NHCO), 10.19 (1H, br s, D₂O exchang., NH–pyrazole). ¹³C-NMR (50 MHz, CDCl₃) δ: 24.8 (cyclohexyl C-3′, C-5′), 25.9 (cyclohexyl C-4′), 33.1 (cyclohexyl C-2′, C-6′), 49.2 (CH₂), 51.2 (cyclohexyl C-1′), 93.0 (C-4), 113.5 (C-2″, C-6″), 119.7 (C-4″), 123.9 (C-3a), 129.6 (C-3″, C-5″), 132.2 (C-7), 134.0 (C-7a), 138.6 (C-3), 146.8 (C-1″), 152.1 (C-5), 169.4 (CO). HR-MS (ESI) m/z: Calcd for C₂₀H₂₅N₆O: [M1+H]⁺=365.2084. Found 365.2090. Anal. Calcd for C₂₀H₂₄N₆O: C, 65.91; H, 6.64; N, 23.06. Found: C, 66.12; H, 6.77; N, 22.89.

2-(Dimethylamino)-N-(5-(phenylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)acetamide (14d)

This compound was prepared according to the general procedure described above, starting from 13j. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (100 : 2, v/v) as the eluent to provide pure 14d as a pale green solid. Yield: 89%, mp: 194°C (EtOAc). ¹H-NMR (400 MHz, DMSO-d₆) δ: 2.33 (6H, s, N(CH₃)₂), 3.16 (2H, s, CH₂), 6.78 (1H, t, H-4′, J=7.2 Hz), 7.20 (3H, m, H-3′, H-5′, H-4), 7.46 (2H, d, H-2′, H-6′, J=7.8 Hz), 8.66 (1H, d, H-7, J=0.8 Hz), 8.69 (1H, s, D₂O exchang., NH–aniline), 10.14 (1H, br s, D₂O exchang., NHCO), 12.87 (1H, s, D₂O exchang., NH–pyrazole). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 45.2 (N(CH₃)₂), 62.1 (CH₂), 98.5 (C-4), 116.4 (C-2′, C-6′), 119.0 (C-4′), 123.0 (C-3a), 128.6 (C-3′, C-5′), 132.0 (C-7), 134.4 (C-7a), 138.6 (C-3), 143.2 (C-1′), 147.7 (C-5), 168.7 (CO). HR-MS (ESI) m/z: Calcd for C₁₆H₁₉N₆O: [M1+H]⁺=311.1615. Found 311.1619. Anal. Calcd for C₁₆H₁₈N₆O: C, 61.92; H, 5.85; N, 27.08. Found: C, 62.11; H, 5.98; N, 27.17.

2-(4-Methylpiperazin-1-yl)-N-(5-(phenylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)acetamide (14e)

This compound was prepared according to the general procedure described above, starting from 13k. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (100 : 5, v/v) as the eluent to provide pure 14e as a green solid. Yield: 78%, mp: 186°C (EtOAc). ¹H-NMR (400 MHz, DMSO-d₆) δ: 2.18 (3H, s, CH₃), 2.40 (4H, br s, piperazine H-3″, H-5″), 2.58 (4H, br s, piperazine H-2″, H-6″), 3.20 (2H, s, CH₂), 6.78 (1H, t, H-4′, J=7.2 Hz), 7.20 (3H, m, H-3′, H-5′, H-4), 7.45 (2H, d, H-2′, H-6′, J=8.0 Hz), 8.66 (1H, s, H-7), 8.70 (1H, s, D₂O exchang., NH–aniline), 10.12 (1H, s, D₂O exchang., NHCO), 12.88 (1H, s, D₂O exchang., NH–pyrazole). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 45.5 (CH₃), 52.5 (piperazine C-2″, C-6″), 54.4 (piperazine C-3″, C-5″), 60.7 (CH₂), 98.5 (C-4), 116.5 (C-2′, C-6′), 119.0 (C-4′), 122.8 (C-3a), 128.5 (C-3′, C-5′), 132.0 (C-7), 134.5 (C-7a), 138.6 (C-3), 143.2 (C-1′), 147.7 (C-5), 168.2 (CO). HR-MS (ESI) m/z: Calcd for C₁₉H₂₄N₇O: [M1+H]⁺=366.2037. Found 366.2044. Anal. Calcd for C₁₉H₂₃N₇O: C, 62.45; H, 6.34; N, 26.83. Found: C, 62.28; H, 6.19; N, 26.99.

2-(Phenylamino)-N-(5-(phenylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)acetamide (14f)

This compound was prepared according to the general procedure described above, starting from 13l. The product was purified by column chromatography (silica gel) using a mixture of cyclohexane–ethyl acetate (1 : 1, v/v) as the eluent to provide pure 14f as a beige solid. Yield: 84%, mp: 126°C (dec.) (EtOAc). ¹H-NMR (400 MHz, CDCl₃) δ: 4.00 (2H, s, CH₂), 4.40 (1H, br s, D₂O exchang., CH₂NH), 6.53 (1H, br s, D₂O exchang., NH–aniline), 6.71 (2H, d, H-2″, H-6″, J=7.8 Hz), 6.85 (1H, t, H-4″, J=7.2 Hz), 7.00 (1H, t, H-4′, J=7.1 Hz), 7.23 (2H, t, H-3″, H-5″, J=7.7 Hz), 7.28 (2H, d, H-2′, H-6′, J=7.7 Hz), 7.33 (2H, t, H-3′, H-5′, J=7.6 Hz), 7.49 (1H, s, H-4), 8.59 (1H, s, H-7), 9.09 (1H, s, D₂O exchang., NHCO), 9.95 (1H, br s, D₂O exchang., NH–pyrazole). ¹³C-NMR (50 MHz, CDCl₃) δ: 49.3 (CH₂), 97.9 (C-4), 113.5 (C-2″, C-6″), 118.4 (C-2′, C-6′), 119.8 (C-4″), 121.7 (C-4′), 123.4 (C-3a), 129.4 (C-3′, C-5′), 129.6 (C-3″, C-5″), 132.4 (C-7), 135.1 (C-7a), 139.8 (C-3), 141.6 (C-1′), 146.6 (C-1″), 148.4 (C-5), 169.3 (CO). HR-MS (ESI) m/z: Calcd for C₂₀H₁₉N₆O: [M1+H]⁺=359.1615. Found 359.1622. Anal. Calcd for C₂₀H₁₈N₆O: C, 67.03; H, 5.06; N, 23.45. Found: C, 66.89; H, 5.01; N, 23.62.

7-Chloro-3-nitro-1H-pyrazolo[3,4-c]pyridine (21)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the nitroderivative 7, upon nitration of pyrazolopyridine 20, in 94% yield. The nitroderivative 21 was obtained pure, as a yellow solid. mp: 172–173°C (EtOAc). ¹H-NMR (600 MHz, DMSO-d₆) δ: 8.03 (1H, d, H-4, J=5.6 Hz), 8.27 (1H, d, H-5, J=5.6 Hz). ¹³C-NMR (151 MHz, DMSO-d₆) δ: 113.9 (C-4), 122.1 (C-3a), 136.4 (C-7a), 137.5 (C-3), 141.5 (C-5), 149.6 (C-7). Anal. Calcd for C₆H₃ClN₄O₂: C, 36.29; H, 1.52; N, 28.22. Found: C, 36.50; H, 1.72; N, 27.94.

7-Chloro-1-methyl-3-nitro-1H-pyrazolo[3,4-c]pyridine (22a) and 7-Chloro-2-methyl-3-nitro-2H-pyrazolo[3,4-c]pyridine (23)

These compounds were prepared following an analogous synthetic procedure to that described for the synthesis of the nitroderivatives 8a and 9a, starting from pyrazolopyridine 21 (540 mg, 2.72 mmol). Purification was effected using a mixture of cyclohexane–ethyl acetate (8 : 2, v/v) as the eluent, to provide the pure isomers 22a (45% yield) and 23 (10% yield).

Data for 22a: Beige solid, mp: 160–161°C (EtOAc). ¹H-NMR (600 MHz, CDCl₃) δ: 4.57 (3H, s, CH₃), 8.08 (1H, d, H-4, J=5.6 Hz), 8.30 (1H, d, H-5, J=5.6 Hz). ¹³C-NMR (151 MHz, CDCl₃) δ: 41.1 (CH₃), 114.5 (C-4), 123.6 (C-3a), 135.2 (C-7a), 135.4 (C-3), 142.5 (C-5), 146.8 (C-7). Anal. Calcd for C₇H₅ClN₄O₂: C, 39.55; H, 2.37; N, 26.35. Found: C, 39.74; H, 2.43; N, 26.17.

Data for 23: Pale yellow solid, mp: 202–203°C (EtOAc). ¹H-NMR (600 MHz, CDCl₃) δ: 4.70 (3H, s, CH₃), 7.93 (1H, d, H-4, J=5.9 Hz), 8.27 (1H, d, H-5, J=5.9 Hz). ¹³C-NMR (151 MHz, CDCl₃) δ: 43.9 (CH₃), 113.2 (C-4), 122.3 (C-3a), 129.0 (C-7a), 140.2 (C-3), 143.5 (C-5), 144.6 (C-7). Anal. Calcd for C₇H₅ClN₄O₂: C, 39.55; H, 2.37; N, 26.35. Found: C, 39.82; H, 2.49; N, 26.21.

7-Chloro-1-(4-methoxybenzyl)-3-nitro-1H-pyrazolo[3,4-c]pyridine (22b)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the derivatives 8b and 9b, starting from pyrazolopyridine 21 (540 mg, 2.72 mmol). Purification was effected using a mixture of cyclohexane–ethyl acetate (8 : 2, v/v) as the eluent, to provide pure 22b as a pale yellow solid, in 65% yield. mp: 142–144°C (CH₂Cl₂/Et₂O). ¹H-NMR (600 MHz, CDCl₃) δ: 3.75 (3H, s, OCH₃), 6.03 (2H, s, CH₂), 6.84 (2H, d, H-2″, H-6″, J=8.5 Hz), 7.29 (2H, d, H-3″, H-5″, J=8.5 Hz), 8.10 (1H, d, H-4, J=5.5 Hz), 8.30 (1H, d, H-5, J=5.8 Hz). ¹³C-NMR (151 MHz, CDCl₃) δ: 55.4 (OCH₃), 55.8 (CH₂), 114.5 (C-2″, C-6″), 114.6 (C-4), 123.9 (C-3a), 126.8 (C-7a), 129.2 (C-3″, C-5″), 134.6 (C-1″), 135.0 (C-3), 142.4 (C-5), 147.4 (C-7), 160.0 (C-4″). Anal. Calcd for C₁₄H₁₁ClN₄O₃: C, 52.76; H, 3.48; N, 17.58. Found: C, 53.02; H, 3.59; N, 17.26.

N-Cyclohexyl-1-methyl-3-nitro-1H-pyrazolo[3,4-c]pyridin-7-amine (24a)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the derivative 10a, upon reaction of the chloroderivative 22a with cyclohexylamine for 2 h, in 89% yield. The product was purified by column chromatography using a mixture of cyclohexane–ethyl acetate (from 7 : 3 up to 6 : 4, v/v) as the eluent, to provide 24a as a yellow solid. mp: 171–173°C (EtOAc/Et₂O). ¹H-NMR (400 MHz, CDCl₃) δ: 1.20–1.31 (3H, m, cyclohexyl H), 1.41–1.50 (2H, m, cyclohexyl H), 1.62–1.68 (1H, m, cyclohexyl H), 1.71–1.78 (2H, m, cyclohexyl H), 2.10–2.15 (2H, m, cyclohexyl H), 4.12–4.24 (1H, m, cyclohexyl H-1′), 4.45 (3H, s, CH₃), 4.78 (1H, br s, D₂O exchang., NH), 7.33 (1H, d, H-4, J=5.8 Hz), 7.95 (1H, d, H-5, J=5.8 Hz). ¹³C-NMR (50 MHz, CDCl₃) δ: 25.0 (cyclohexyl C-3′, C-5′), 25.9 (cyclohexyl C-4′), 33.4 (cyclohexyl C-2′, C-6′), 41.1 (CH₃), 49.8 (cyclohexyl C-1′), 104.0 (C-4), 123.0 (C-3a), 128.8 (C-7a), 142.7 (C-5), 145.2 (C-3), 147.0 (C-7). Anal. Calcd for C₁₃H₁₇N₅O₂: C, 56.72; H, 6.22; N, 25.44. Found: C, 56.93; H, 6.34; N, 25.29.

N-Cyclohexyl-1-(4-methoxybenzyl)-3-nitro-1H-pyrazolo[3,4-c]pyridin-7-amine (24b)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the derivative 10a, upon reaction of the chloroderivative 22b with cyclohexylamine for 3 h, in 70% yield. The product was purified by column chromatography using a mixture of cyclohexane–ethyl acetate (90 : 10, v/v) as the eluent, to provide 24b as an orange colored solid. mp: 152–154°C (CH₂Cl₂/Et₂O). ¹H-NMR (600 MHz, CDCl₃) δ: 0.82–0.92 (3H, m, cyclohexyl H), 1.08–1.18 (2H, m, cyclohexyl H), 1.30–1.40 (1H, m, cyclohexyl H), 1.57–1.65 (2H, m, cyclohexyl H), 1.82–1.88 (2H, m, cyclohexyl H), 3.81 (3H, s, OCH₃), 3.88–3.96 (1H, m, cyclohexyl H-1′), 4.64 (1H, d, D₂O exchang., NH–cyclohexylamine, J=6.4 Hz), 5.82 (2H, s, CH₂), 6.94 (2H, d, H-2″, H-6″, J=8.7 Hz), 7.09 (2H, d, H-3″, H-5″, J=8.7 Hz), 7.36 (1H, d, H-4, J=5.8 Hz), 7.94 (1H, d, H-5, J=5.8 Hz). ¹³C-NMR (151 MHz, CDCl₃) δ: 25.1 (cyclohexyl C-3′, C-5′), 25.9 (cyclohexyl C-4′), 33.4 (cyclohexyl C-2′, C-6′), 49.4 (cyclohexyl C-1′), 55.4 (OCH₃), 57.7 (CH₂), 101.7 (C-4), 114.4 (C-2″, C-6″), 122.4 (C-3a), 126.6 (C-7a), 129.7 (C-3″, C-5″), 134.7 (C-1″), 137.0 (C-3), 145.4 (C-5), 150.2 (C-7), 160.0 (C-4″). Anal. Calcd for C₂₀H₂₃N₅O₃: C, 62.98; H, 6.08; N, 18.36. Found: C, 62.78; H, 6.01; N, 18.55.

N⁷-Cyclohexyl-1-methyl-1H-pyrazolo[3,4-c]pyridine-3,7-diamine (25a)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the aminoderivative 11a, starting from the nitroderivative 24a (600 mg, 2.18 mmol), in 82% yield. The product was purified by column chromatography using a mixture of cyclohexane–ethyl acetate (from 6 : 4 up to 3 : 7, v/v) as the eluent, to provide pure 25a as a beige solid. mp: 144–146°C (CH₂Cl₂/petroleum ether). ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.15–1.25 (3H, m, cyclohexyl H), 1.30–1.40 (2H, m, cyclohexyl H), 1.58–1.65 (1H, m, cyclohexyl H), 1.70–1.78 (2H, m, cyclohexyl H), 1.96–2.10 (2H, m, cyclohexyl H), 3.92 (3H, s, CH₃), 3.93–3.98 (1H, m, cyclohexyl H-1′), 5.27 (2H, br s, D₂O exchang., NH₂), 5.62 (1H, d, D₂O exchang., NH, J=7.3 Hz), 6.82 (1H, d, H-4, J=5.7 Hz), 7.44 (1H, d, H-5, J=5.7 Hz). ¹³C-NMR (151 MHz, DMSO-d₆) δ: 24.9 (cyclohexyl C-3′, C-5′), 25.7 (cyclohexyl C-4′), 32.3 (cyclohexyl C-2′, C-6′), 37.8 (CH₃), 49.0 (cyclohexyl C-1′), 104.0 (C-4), 118.2 (C-3a), 128.3 (C-7a), 134.5 (C-5), 144.9 (C-3), 148.0 (C-7). HR-MS (ESI) m/z: Calcd for C₁₃H₂₀N₅: [M1+H]⁺=246.1713. Found 246.1715. Anal. Calcd for C₁₃H₁₉N₅: C, 63.65; H, 7.81; N, 28.55. Found: C, 63.90; H, 8.01; N, 28.09.

N⁷-Cyclohexyl-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-c]pyridine-3,7-diamine (26a)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the aminoderivative 11a, starting from the nitroderivative 24b (720 mg, 1.89 mmol), in 80% yield. The product was purified by column chromatography using a mixture of cyclohexane–ethyl acetate (from 1 : 1 up to 3 : 7, v/v) as the eluent, to provide pure 26a as a grey solid. mp: 130–132°C (CH₂Cl₂/n-hexane). ¹H-NMR (400 MHz, DMSO-d₆) δ: 1.10–1.25 (3H, m, cyclohexyl H), 1.30–1.40 (2H, m, cyclohexyl H), 1.52–1.65 (1H, m, cyclohexyl H), 1.66–1.74 (2H, m, cyclohexyl H), 1.86–1.94 (2H, m, cyclohexyl H), 3.70 (3H, s, OCH₃), 3.94 (1H, br s, cyclohexyl H-1′), 5.31 (2H, s, CH₂), 6.14 (2H, br s, D₂O exchang., NH₂), 6.31 (1H, br s, D₂O exchang., NH), 6.69 (1H, d, H-4, J=6.1 Hz), 6.88 (2H, d, H-2″, H-6″, J=8.5 Hz), 7.06–7.16 (3H, m, H-5, H-3″, H-5″). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 25.0 (cyclohexyl C-3′, C-5′), 25.3 (cyclohexyl C-4′), 32.5 (cyclohexyl C-2′, C-6′), 48.6 (cyclohexyl C-1′), 50.3 (CH₂), 55.1 (OCH₃), 103.0 (C-4), 108.4 (C-3a), 113.8 (C-2″, C-6″), 128.6 (C-3″, C-5″), 128.9 (C-7a, C-1″), 135.2 (C-5), 141.0 (C-3), 147.8 (C-7), 158.6 (C-4″). Anal. Calcd for C₂₀H₂₅N₅O: C, 68.35; H, 7.17; N, 19.93. Found: C, 68.21; H, 7.10; N, 20.12.

2-Chloro-N-(7-(cyclohexylamino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-3-yl)acetamide (25b) and 2-Chloro-N-(7-(cyclohexylamino)-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-c]pyridin-3-yl)acetamide (26b)

These compounds were prepared following an analogous synthetic procedure to that described for the synthesis of the compounds 12a and c, starting from the amines 25a and 26a, respectively. These intermediates were used imediately to the next step, with no further purification.

N-(7-(Cyclohexylamino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(dimethylamino)acetamide (27a)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the compounds 13a–l, upon reaction of the chloride 25b with dimethylamine (5.6 M solution in ethanol). The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (from 100 : 5 up to 100 : 15, v/v) as the eluent to provide pure 27a as a beige solid. Yield: 97%, mp: 145–147°C (Et₂O). ¹H-NMR (600 MHz, CDCl₃) δ: 1.21–1.29 (3H, m, cyclohexyl H), 1.43–1.51 (2H, m, cyclohexyl H), 1.61–1.67 (1H, m, cyclohexyl H), 1.70–1.76 (2H, m, cyclohexyl H), 2.09–2.15 (2H, m, cyclohexyl H), 2.40 (6H, s, N(CH₃)₂), 3.15 (2H, s, CH₂), 4.06–4.14 (1H, m, cyclohexyl H-1′), 4.22 (3H, s, CH₃– pyrazole), 4.64 (1H, d, D₂O exchang., NH–cyclohexylamine, J=6.1 Hz), 7.00 (1H, d, H-4, J=5.9 Hz), 7.63 (1H, d, H-5, J=5.9 Hz), 9.43 (1H, br s, D₂O exchang., NHCO). ¹³C-NMR (151 MHz, CDCl₃) δ: 24.9 (cyclohexyl C-3′, C-5′), 25.9 (cyclohexyl C-4′), 33.4 (cyclohexyl C-2′, C-6′), 39.2 (CH₃–pyrazole), 46.1 (N(CH₃)₂), 49.4 (cyclohexyl C-1′), 63.1 (CH₂), 105.6 (C-4), 121.7 (C-3a), 128.4 (C-7a), 136.7 (C-5), 138.0 (C-3), 145.1 (C-7), 168.9 (CO). HR-MS (ESI) m/z: Calcd for C₁₇H₂₇N₆O: [M1+H]⁺=331.2241. Found 331.2247. Anal. Calcd for C₁₇H₂₆N₆O: C, 61.79; H, 7.93; N, 25.43. Found: C, 61.96; H, 7.99; N, 25.31.

N-(7-(Cyclohexylamino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(4-methylpiperazin-1-yl)acetamide (27b)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the compounds 13a–l, upon reaction of the chloride 25b with 1-methylpiperazine. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (from 100 : 5 up to 100 : 20, v/v) as the eluent to provide pure 27b as a yellow oil. Yield: 96%. ¹H-NMR (600 MHz, CDCl₃) δ: 1.20–1.30 (3H, m, cyclohexyl H), 1.40–1.50 (2H, m, cyclohexyl H), 1.60–1.68 (1H, m, cyclohexyl H), 1.69–1.77 (2H, m, cyclohexyl H), 2.05–2.15 (2H, m, cyclohexyl H), 2.32 (3H, s, CH₃–piperazine), 2.50–2.60 (4H, br s, piperazine H-3″, H-5″), 2.65–2.80 (4H, br s, piperazine H-2″, H-6″), 3.19 (2H, s, CH₂), 4.06–4.14 (1H, m, cyclohexyl H-1′), 4.24 (3H, s, CH₃–pyrazole), 4.60 (1H, d, D₂O exchang., NH–cyclohexylamine, J=7.0 Hz), 6.94 (1H, d, H-4, J=5.9 Hz), 7.65 (1H, d, H-5, J=5.9 Hz), 9.31 (1H, br s, D₂O exchang., NHCO). ¹³C-NMR (151 MHz, CDCl₃) δ: 24.9 (cyclohexyl C-3′, C-5′), 26.0 (cyclohexyl C-4′), 33.5 (cyclohexyl C-2′, C-6′), 39.2 (CH₃–pyrazole), 45.9 (CH₃–piperazine), 49.3 (cyclohexyl C-1′), 53.4 (piperazine C-2″, C-6″), 55.1 (piperazine C-3″, C-5″), 61.5 (CH₂), 105.4 (C-4), 121.7 (C-3a), 128.4 (C-7a), 137.1 (C-5), 137.8 (C-3), 145.2 (C-7), 168.5 (CO). HR-MS (ESI) m/z: Calcd for C₂₀H₃₂N₇O: [M1+H]⁺=386.2663. Found 386.2665. Anal. Calcd for C₂₀H₃₁N₇O: C, 62.31; H, 8.11; N, 25.43. Found: C, 62.63; H, 8.29; N, 25.21.

N-(7-(Cyclohexylamino)-1-methyl-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(phenylamino)acetamide (27c)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the compounds 13a–l, upon reaction of the chloride 25b with aniline. The product was purified by column chromatography (silica gel) using a mixture of chloroform–methanol (100 : 2, v/v) as the eluent to provide pure 27c as a pale yellow solid. Yield: 51%. mp: >300°C (EtOAc). ¹H-NMR (600 MHz, CDCl₃) δ: 1.20–1.30 (3H, m, cyclohexyl H), 1.42–1.52 (2H, m, cyclohexyl H), 1.60–1.68 (1H, m, cyclohexyl H), 1.69–1.78 (2H, m, cyclohexyl H), 2.06–2.14 (2H, m, cyclohexyl H), 3.94 (2H, s, CH₂), 4.06–4.14 (1H, m, cyclohexyl H-1′), 4.16 (3H, s, CH₃), 4.62 (1H, br s, D₂O exchang., NH–cyclohexylamine), 4.83 (1H, br s, D₂O exchang., NH–aniline), 6.67 (2H, d, H-2″, H-6″, J=7.9 Hz), 6.81 (1H, t, H-4″, J=7.3 Hz), 6.91 (1H, d, H-4, J=5.9 Hz), 7.20 (2H, t, H-3″, H-5″, J=7.8 Hz), 7.58 (1H, d, H-5, J=5.9 Hz), 9.06 (1H, br s, D₂O exchang., NHCO). ¹³C-NMR (151 MHz, CDCl₃) δ: 24.9 (cyclohexyl C-3′, C-5′), 25.9 (cyclohexyl C-4′), 33.3 (cyclohexyl C-2′, C-6′), 39.3 (CH₃), 49.2 (CH₂), 49.8 (cyclohexyl C-1′), 105.4 (C-4), 113.5 (C-2″, C-6″), 119.4 (C-4″), 122.0 (C-3a), 128.1 (C-7a), 129.6 (C-3″, C-5″), 135.7 (C-5), 137.6 (C-3), 144.8 (C-1″), 147.1 (C-7), 169.7 (CO). HR-MS (ESI) m/z: Calcd for C₂₁H₂₇N₆O: [M1+H]⁺=379.2241. Found 379.2245. Anal. Calcd for C₂₁H₂₆N₆O: C, 66.64; H, 6.92; N, 22.20. Found: C, 66.87; H, 7.00; N, 22.11.

N-(7-(Cyclohexylamino)-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(dimethylamino)acetamide (28)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the compounds 13a–l, upon reaction of the chloride 26b with dimethylamine (5.6 M solution in ethanol). The product was purified by column chromatography (silica gel) using a mixture of ethyl acetate–methanol (from 100 : 5 up to 100 : 10, v/v) as the eluent to provide pure 28 as a white solid. Yield: 76%, mp: 138–139°C (Et₂O). ¹H-NMR (600 MHz, CDCl₃) δ: 0.78–0.88 (3H, m, cyclohexyl H), 1.00–1.10 (2H, m, cyclohexyl H), 1.26–1.35 (1H, m, cyclohexyl H), 1.47–1.58 (2H, m, cyclohexyl H), 1.77–1.85 (2H, m, cyclohexyl H), 2.39 (6H, s, N(CH₃)₂), 3.14 (2H, s, NHCOCH₂), 3.75 (3H, s, OCH₃), 3.82–3.90 (1H, m, cyclohexyl H-1″), 4.41 (1H, d, D₂O exchang., NH–cyclohexylamine, J=7.2 Hz), 5.61 (2H, s, CH₂–pyrazole), 6.85 (2H, d, H-2′, H-6′, J=8.7 Hz), 7.01–7.06 (3H, m, H-4, H-3′, H-5′), 7.64 (1H, d, H-5, J=5.9 Hz), 9.51 (1H, br s, D₂O exchang., NHCO). ¹³C-NMR (151 MHz, CDCl₃) δ: 24.7 (cyclohexyl C-3″, C-5″), 25.86 (cyclohexyl C-4″), 33.0 (cyclohexyl C-2″, C-6″), 46.1 (N(CH₃)₂), 49.1 (cyclohexyl C-1″), 55.1 (CH₂–pyrazole), 55.4 (OCH₃), 63.2 (NHCOCH₂), 105.5 (C-4), 114.8 (C-2′, C-6′), 122.2 (C-3a), 127.5 (C-3′, C-5′), 128.2 (C-7a), 128.9 (C-1′), 137.2 (C-5), 138.4 (C-3), 145.0 (C-7), 159.8 (C-4′), 168.9 (CO). HR-MS (ESI) m/z: Calcd for C₂₄H₃₃N₆O₂: [M1+H]⁺=437.2660. Found 437.2668. Anal. Calcd for C₂₄H₃₂N₆O₂: C, 66.03; H, 7.39; N, 19.25. Found: C, 66.33; H, 7.54; N, 18.98.

N-(7-(Cyclohexylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(dimethylamino)acetamide (29)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the compounds 14a–f, starting from the derivative 28. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (9 : 1, v/v) as the eluent to provide pure 29 as a colorless oil. Yield: 99%. ¹H-NMR (400 MHz, DMSO-d₆) δ: 1.15–1.18 (3H, m, cyclohexyl H), 1.21–1.42 (2H, m, cyclohexyl H), 1.58–1.64 (1H, m, cyclohexyl H), 1.71–1.79 (2H, m, cyclohexyl H), 1.97–2.05 (2H, m, cyclohexyl H), 2.35 (6H, s, N(CH₃)₂), 3.21 (2H, s, CH₂), 3.93–4.05 (1H, m, cyclohexyl H-1′), 6.55 (1H, br s, D₂O exchang., NH–cyclohexylamine), 6.80 (1H, d, H-4, J=5.9 Hz), 7.49 (1H, d, H-5, J=5.9 Hz), 10.11 (1H, br s, D₂O exchang., NHCO), 12.77 (1H, br s, D₂O exchang., NH–pyrazole). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 24.6 (cyclohexyl C-3′, C-5′), 25.5 (cyclohexyl C-4′), 32.7 (cyclohexyl C-2′, C-6′), 45.1 (N(CH₃)₂), 48.4 (cyclohexyl C-1′), 61.9 (CH₂), 104.0 (C-4), 119.0 (C-3a), 127.8 (C-7a), 136.0 (C-5), 139.8 (C-3), 144.4 (C-7), 168.2 (CO). HR-MS (ESI) m/z: Calcd for C₁₆H₂₅N₆O: [M1+H]⁺=317.2084. Found 317.2087. Anal. Calcd for C₁₆H₂₄N₆O: C, 60.74; H, 7.65; N, 26.56. Found: C, 60.43; H, 7.52; N, 26.75.

1-Methyl-3-nitro-N-phenyl-1H-pyrazolo[3,4-c]pyridin-7-amine (30)

Aniline (0.17 mL, 1.87 mmol) was added into a solution of the chlorocompound 22a (80 mg, 0.37 mmol) in 2-ethoxyethanol (2 mL), under argon, and the resulting solution was heated at 130°C for 3h. The solvent was then vacuum-evaporated and the residue was purified by column chromatography (silica gel) using dichloromethane as the eluent, to give pure 30 (60 mg, 59%) as a yellow solid. mp: 197–198°C (EtOAc). ¹H-NMR (600 MHz, CDCl₃) δ: 4.34 (3H, s, CH₃), 7.14 (1H, t, H-4′, J=7.3 Hz), 7.28 (2H, d, H-2′, H-6′, J=7.6 Hz), 7.36 (2H, t, H-3′, H-5′, J=7.5 Hz), 7.57 (1H, br s, H-4), 7.88 (1H, br s, H-5). ¹³C-NMR (151 MHz, CDCl₃) δ: 41.4 (CH₃), 107.7 (C-4), 119.6 (C-3a), 120.7 (C-2′, C-6′), 124.6 (C-4′), 129.9 (C-3′, C-5′), 130.6 (C-7a), 134.9 (C-5), 140.9 (C-3), 142.6 (C-1′), 147.3 (C-7). Anal. Calcd for C₁₃H₁₁N₅O₂: C, 57.99; H, 4.12; N, 26.01. Found: C, 58.14; H, 4.22; N, 25.84.

1-Methyl-N⁷-phenyl-1H-pyrazolo[3,4-c]pyridine-3,7-diamine (31)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the compound 11a, upon hydrogenation of the nitroderivative 30. The product was purified by column chromatography (silica gel) using a mixture of cyclohexane–ethyl acetate (from 5 : 5 up to 2 : 8, v/v) as the eluent to provide pure 31 as an orange colored solid. Yield: 75%, mp: 120–122°C (CH₂Cl₂/n-hexane). ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.93 (3H, s, CH₃), 5.52 (2H, br s, D₂O exchang., NH₂), 6.90 (1H, t, H-4′, J=7.3 Hz), 7.20 (1H, d, H-4, J=5.6 Hz), 7.25 (2H, t, H-3′, H-5′, J=7.8 Hz), 7.44 (2H, d, H-2′, H-6′, J=7.8 Hz), 7.60 (1H, d, H-5, J=5.6 Hz), 8.37 (1H, br s, D₂O exchang., NH). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 37.6 (CH₃), 108.3 (C-4), 118.8 (C-2′, C-6′), 119.7 (C-3a), 120.6 (C-4′), 128.4 (C-3′, C-5′), 129.8 (C-7a), 134.0 (C-5), 141.6 (C-3), 142.2 (C-1′), 148.4 (C-7). Anal. Calcd for C₁₃H₁₃N₅: C, 65.25; H, 5.48; N, 29.27. Found: C, 65.37; H, 5.60; N, 29.03.

2-Chloro-N-(1-methyl-7-(phenylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)acetamide (32)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the compounds 12b and d, starting from the amine 31. This intermediate was used imediately to the next step, with no further purification.

2-(Dimethylamino)-N-(1-methyl-7-(phenylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)acetamide (33a)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the compounds 13a–l, upon reaction of the chloride 32 with dimethylamine (5.6 M solution in ethanol). The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (from 100 : 0.5 up to 100 : 5, v/v) as the eluent to provide pure 33a as a beige solid, in 97% yield. mp: 140–142°C (EtOH/Et₂O). ¹H-NMR (600 MHz, CDCl₃) δ: 2.44 (6H, s, N(CH₃)₂), 3.19 (2H, s, CH₂), 4.09 (3H, s, CH₃–pyrazole), 6.77 (1H, br s, D₂O exchang., NH–aniline), 7.02 (1H, t, H-4′, J=7.3 Hz), 7.21 (2H, d, H-2′, H-6′, J=7.6 Hz), 7.29 (2H, t, H-3′, H-5′, J=7.7 Hz), 7.45 (1H, br s, H-4), 7.79 (1H, br s, H-5), 9.62 (1H, br s, D₂O exchang., NHCO). ¹³C-NMR (151 MHz, CDCl₃) δ: 38.9 (CH₃–pyrazole), 46.1 (N(CH₃)₂), 63.1 (CH₂), 111.0 (C-4), 119.2 (C-2′, C-6′), 122.7 (C-3a, C-4′), 129.5 (C-3′, C-5′), 130.6 (C-7a), 136.6 (C-5), 138.7 (C-3), 141.9 (C-1′), 142.3 (C-7), 168.9 (CO). HR-MS (ESI) m/z: Calcd for C₁₇H₂₁N₆O: [M1+H]⁺=325.1771. Found 325.1773. Anal. Calcd for C₁₇H₂₀N₆O: C, 62.95; H, 6.21; N, 25.91. Found: C, 63.11; H, 6.28; N, 25.72.

N-(1-Methyl-7-(phenylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(4-methylpiperazin-1-yl)acetamide (33b)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the compounds 13a–l, upon reaction of the chloride 32 with 1-methylpiperazine. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (from 100 : 2 up to 100 : 10, v/v) as the eluent to provide pure 33b as a white solid, in 99% yield. mp: 202–204°C (EtOH/Et₂O). ¹H-NMR (600 MHz, CDCl₃) δ: 2.34 (3H, s, CH₃–piperazine), 2.50–2.60 (4H, br s, piperazine H-3″, H-5″), 2.65–2.80 (4H, br s, piperazine H-2″, H-6″), 3.23 (2H, s, CH₂), 4.10 (3H, s, CH₃–pyrazole), 6.58 (1H, br s, D₂O exchang., NH–aniline), 7.03 (1H, t, H-4′, J=7.3 Hz), 7.22 (2H, d, H-2′, H-6′, J=7.8 Hz), 7.30 (2H, t, H-3′, H-5′, J=7.9 Hz), 7.46 (1H, d, H-4, J=5.8 Hz), 7.85 (1H, d, H-5, J=5.8 Hz), 9.49 (1H, br s, D₂O exchang., NHCO). ¹³C-NMR (151 MHz, CDCl₃) δ: 38.9 (CH₃–pyrazole), 46.1 (CH₃–piperazine), 53.7 (piperazine C-2″, C-6″), 55.3 (piperazine C-3″, C-5″), 61.7 (CH₂), 111.1 (C-4), 119.1 (C-2′, C-6′), 122.7 (C-4′), 122.8 (C-3a), 129.5 (C-3′, C-5′), 130.6 (C-7a), 136.9 (C-5), 138.7 (C-3), 141.8 (C-7), 142.1 (C-1′), 168.7 (CO). HR-MS (ESI) m/z: Calcd for C₂₀H₂₆N₇O: [M1+H]⁺=380.2193. Found 380.2198. Anal. Calcd for C₂₀H₂₅N₇O: C, 63.30; H, 6.64; N, 25.84. Found: C, 63.55; H, 6.84; N, 25.59.

N-(1-Methyl-7-(phenylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)-2-(phenylamino)acetamide (33c)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the compounds 13a–l, upon reaction of the chloride 32 with aniline. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–ethyl acetate (1 : 1, v/v) as the eluent to provide pure 33c as a beige solid, in 55% yield. mp: 180–181°C (EtOH/Et₂O). ¹H-NMR (400 MHz, DMSO-d₆) δ: 3.94 (2H, s, CH₂), 4.22 (3H, s, CH₃), 6.05 (1H, m, D₂O exchang., NHCH₂), 6.56–6.65 (3H, m, H-2″, H-4″, H-6″), 6.94 (1H, t, H-4′, J=7.4 Hz), 7.09–7.15 (3H, m, H-4, H-3′, H-5′), 7.28 (2H, t, H-3″, H-5″, J=7.9 Hz), 7.48 (2H, d, H-2′, H-6′, J=8.1 Hz), 7.65 (1H, d, H-5, J=5.7 Hz), 8.48 (1H, br s, D₂O exchang., NH–aniline), 10.45 (1H, br s, D₂O exchang., NHCO). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 38.8 (CH₃), 46.6 (CH₂), 109.4 (C-4), 112.3 (C-2″, C-6″), 116.5 (C-4″), 119.4 (C-2′, C-6′), 121.2 (C-4′), 122.5 (C-3a), 128.5 (C-3″, C-5″), 128.9 (C-3′, C-5′), 129.4 (C-7a), 135.4 (C-5), 138.4 (C-3), 141.7 (C-1″), 142.3 (C-7), 148.3 (C-1′), 169.7 (CO). HR-MS (ESI) m/z: Calcd for C₂₁H₂₁N₆O: [M1+H]⁺=373.1771. Found 373.1774. Anal. Calcd for C₂₁H₂₀N₆O: C, 67.73; H, 5.41; N, 22.57. Found: C, 67.81; H, 5.44; N, 22.47.

3-Nitro-N-phenyl-1H-pyrazolo[3,4-c]pyridin-7-amine (34)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of 30, upon treatment of 21 with aniline in 1,4-dioxane, at 100°C for 12h. The product was purified by column chromatography (silica gel) using a mixture of dichloromethane–methanol (98 : 2, v/v) as the eluent, to provide pure 34, in 95% yield as a yellow solid. mp: 260–262°C (EtOAc). ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.11 (1H, t, H-4′, J=7.1 Hz), 7.38–7.44 (3H, m, H-4, H-3′, H-5′), 7.82 (2H, d, H-2′, H-6′, J=7.8 Hz), 7.95 (1H, br s, H-5), 9.30 (1H, br s, D₂O exchang., NH–aniline). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 104.6 (C-4), 120.0 (C-2′, C-6′), 120.5 (C-3a), 123.2 (C-4′), 129.1 (C-3′, C-5′), 130.1 (C-7a), 139.2 (C-3, C-1′), 142.8 (C-5), 148.8 (C-7). Anal. Calcd for C₁₂H₉N₅O₂: C, 56.47; H, 3.55; N, 27.44. Found: C, 56.66; H, 3.61; N, 27.31.

2-(4-Methoxybenzyl)-3-nitro-N-phenyl-2H-pyrazolo[3,4-c]pyridin-7-amine (35)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the compound 22b. The product was purified by column chromatography (silica gel) using a mixture of cyclohexane–ethyl acetate–dichloromethane (from 90 : 10 : 3 up to 85 : 15 : 4, v/v/v) as the eluent, to provide pure 34 as an orange colored solid, in 45% yield. mp: 175–177°C (MeOH). ¹H-NMR (600 MHz, CDCl₃) δ: 3.78 (3H, s, OCH₃), 6.04 (2H, s, CH₂), 6.87 (2H, d, H-2″, H-6″, J=8.6 Hz), 7.12 (1H, t, H-4′, J=7.3 Hz), 7.29 (1H, d, H-4, J=6.0 Hz), 7.35 (2H, d, H-3″, H-5″, J=8.6 Hz), 7.41 (2H, t, H-3′, H-5′, J=7.8 Hz), 7.70 (1H, br s, D₂O exchang., NH), 7.89 (2H, d, H-2′, H-6′, J=7.8 Hz), 8.05 (1H, d, H-5, J=6.0 Hz). ¹³C-NMR (151 MHz, CDCl₃) δ: 55.5 (OCH₃), 58.0 (CH₂), 104.5 (C-4), 114.5 (C-2″, C-6″), 119.9 (C-2′, C-6′), 122.2 (C-3a), 123.4 (C-4′), 126.4 (C-7a), 129.2 (C-3′, C-5′), 129.9 (C-3″, C-5″), 134.7 (C-1″), 137.3 (C-3), 139.1 (C-1′), 144.7 (C-5), 147.8 (C-7), 160.2 (C-4″). Anal. Calcd for C₂₀H₁₇N₅O₃: C, 63.99; H, 4.56; N, 18.66. Found: C, 64.34; H, 4.87; N, 18.33.

2-(4-Methoxybenzyl)-N⁷-phenyl-2H-pyrazolo[3,4-c]pyridine-3,7-diamine (36)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the compound 11a, upon hydrogenation of the nitroderivative 34. The product was purified by column chromatography (silica gel) using a mixture of cyclohexane–ethyl acetate (from 7 : 3 up to 5 : 5, v/v) as the eluent to provide pure 35 as a brown solid, in 96% yield. mp: 54–56°C (CH₂Cl₂/n-hexane). ¹H-NMR (600 MHz, DMSO-d₆) δ: 3.71 (3H, s, OCH₃), 5.40 (2H, s, CH₂), 6.23 (2H, br s, D₂O exchang., NH₂), 6.87–6.91 (3H, m, H-4′, H-2″, H-6″), 6.95 (1H, d, H-4, J=5.9 Hz), 7.17 (2H, d, H-3″, H-5″, J=8.5 Hz), 7.25 (2H, t, H-3′, H-5′, J=7.8 Hz), 7.31 (1H, d, H-5, J=5.9 Hz), 8.04 (2H, d, H-2′, H-6′, J=7.7 Hz), 8.57 (1H, br s, D₂O exchang., NH). ¹³C-NMR (151 MHz, DMSO-d₆) δ: 50.4 (CH₂), 55.1 (OCH₃), 106.0 (C-4), 109.3 (C-3a), 113.8 (C-2″, C-6″), 118.9 (C-2′, C-6′), 120.7 (C-4′), 128.2 (C-3′, C-5′, C-7a), 128.6 (C-3″, C-5″), 128.9 (C-1″), 131.0 (C-5), 135.8 (C-3), 141.3 (C-1′), 145.8 (C-7), 158.6 (C-4″). Anal. Calcd for C₂₀H₁₉N₅O: C, 69.55; H, 5.54; N, 20.28. Found: C, 69.23; H, 5.31; N, 20.49.

2-Chloro-N-(2-(4-methoxybenzyl)-7-(phenylamino)-2H-pyrazolo[3,4-c]pyridin-3-yl)acetamide (37)

This compound was prepared following an analogous synthetic procedure to that described for the synthesis of the compounds 12b and d, starting from the amine 36. This intermediate was used imediately to the next step, with no further purification.

2-(Dimethylamino)-N-(2-(4-methoxybenzyl)-7-(phenylamino)-2H-pyrazolo[3,4-c]pyridin-3-yl)acetamide (38a) and N-(2-(4-Methoxybenzyl)-7-(phenylamino)-2H-pyrazolo[3,4-c]pyridin-3-yl)-2-(4-methylpiperazin-1-yl)acetamide (38b)

These compounds were prepared following an analogous synthetic procedure to that described for the synthesis of the compounds 13a–l, upon reaction of the chloride 37 with dimethylamine (5.6 M solution in ethanol) or 1-methylpiperazine, respectively. The product was purified by column chromatography using a mixture of dichloromethane–methanol (95 : 5, v/v) as the eluent to provide pure 38a or b.

Data for 38a: Yield 98%. Beige solid, mp: 183–185°C (CH₂Cl₂/Et₂O). ¹H-NMR (600 MHz, CDCl₃) δ: 2.32 (6H, s, N(CH₃)₂), 3.11 (2H, s, NHCOCH₂), 3.76 (3H, s, OCH₃), 5.45 (2H, s, CH₂–pyrazole), 6.83–6.87 (3H, m, H-4, H-2″, H-6″), 7.02 (1H, t, H-4′, J=7.3 Hz), 7.12 (2H, d, H-3″, H-5″, J=8.5 Hz), 7.35 (2H, t, H-3′, H-5′, J=7.8 Hz), 7.68 (1H, d, H-5, J=6.1 Hz), 7.87 (2H, d, H-2′, H-6′, J=7.9 Hz), 8.70–9.40 (1H, br s, D₂O exchang., NHCO). ¹³C-NMR (151 MHz, CDCl₃) δ: 46.1 (N(CH₃)₂), 54.3 (CH₂–pyrazole), 55.4 (OCH₃), 62.8 (NHCOCH₂), 104.7 (C-4), 114.4 (C-2″, C-6″), 118.1 (C-3a), 119.4 (C-2′, C-6′), 122.2 (C-4′), 127.1 (C-7a), 128.0 (C-1″), 128.7 (C-3″, C-5″), 129.0 (C-3′, C-5′), 136.1 (C-3), 136.6 (C-5), 140.1 (C-1′), 147.3 (C-7), 159.7 (C-4″), 169.5 (CO). HR-MS (ESI) m/z: Calcd for C₂₄H₂₇N₆O₂: [M1+H]⁺=431.2190. Found 431.2198. Anal. Calcd for C₂₄H₂₆N₆O₂: C, 66.96; H, 6.09; N, 19.52. Found: C, 67.17; H, 6.18; N, 19.44.

Data for 38b: Yield 97%. White solid, mp: 159–160°C (EtOAc/Et₂O). ¹H-NMR (600 MHz, CDCl₃) δ: 2.27 (3H, s, CH₃), 2.30–2.40 (4H, br s, piperazine H-3″′, H-5‴), 2.55–2.59 (4H, br s, piperazine H-2″′, H-6‴), 3.17 (2H, s, NHCOCH₂), 3.77 (3H, s, OCH₃), 5.49 (2H, s, CH₂–pyrazole), 6.79 (1H, d, H-4, J=6.1 Hz), 6.86 (2H, d, H-2″, H-6″, J=8.7 Hz), 7.02 (1H, t, H-4′, J=7.4 Hz), 7.07 (2H, d, H-3″, H-5″, J=8.7 Hz), 7.35 (2H, t, H-3′, H-5′, J=8.0 Hz), 7.58 (1H, br s, D₂O exchang., NH–aniline), 7.70 (1H, d, H-5, J=6.1 Hz), 7.88 (2H, d, H-2′, H-6′, J=7.6 Hz), 9.00 (1H, br s, D₂O exchang., NHCO). ¹³C-NMR (151 MHz, CDCl₃) δ: 46.0 (CH₃), 53.6 (piperazine C-2″′, C-6‴), 54.3 (CH₂–pyrazole), 55.0 (piperazine C-3″′, C-5‴), 55.4 (OCH₃), 61.3 (NHCOCH₂), 104.4 (C-4), 114.6 (C-2″, C-6″), 118.2 (C-3a), 119.3 (C-2′, C-6′), 122.3 (C-4′), 127.3 (C-7a), 127.9 (C-1″), 128.3 (C-3″, C-5″), 129.0 (C-3′, C-5′), 136.2 (C-3), 137.0 (C-5), 140.1 (C-1′), 147.3 (C-7), 159.7 (C-4″), 169.0 (CO). HR-MS (ESI) m/z: Calcd for C₂₇H₃₂N₇O₂: [M1+H]⁺=486.2612. Found 486.2616. Anal. Calcd for C₂₇H₃₁N₇O₂: C, 66.78; H, 6.43; N, 20.19. Found: C, 67.05; H, 6.54; N, 19.98.

2-(Dimethylamino)-N-(7-(phenylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)acetamide (39a) and 2-(4-Methylpiperazin-1-yl)-N-(7-(phenylamino)-1H-pyrazolo[3,4-c]pyridin-3-yl)acetamide (39b)

These compounds were prepared following an analogous synthetic procedure to that described for the synthesis of the compounds 14a–f, upon reaction of the derivatives 38a and b with trifluoroacetic acid, respectively. The product was purified by column chromatography using a mixture of dichloromethane–methanol (9 : 1, v/v) as the eluent to provide pure 39a or b.

• Data for 39a: Yield 92%. Beige gum. ¹H-NMR (400 MHz, DMSO-d₆) δ: 2.32 (6H, s, N(CH₃)₂), 3.16 (2H, s, CH₂), 6.97 (1H, t, H-4′, J=7.3 Hz), 7.12 (1H, d, H-4, J=5.8 Hz), 7.34 (2H, t, H-3′, H-5′, J=8.0 Hz), 7.69 (1H, d, H-5, J=5.8 Hz), 7.89 (2H, d, H-2′, H-6′, J=8.0 Hz), 8.95 (1H, br s, D₂O exchang., NH–aniline), 10.17 (1H, br s, D₂O exchang., NHCO), 12.90 (1H, br s, D₂O exchang., NH–pyrazole). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 45.3 (N(CH₃)₂), 62.2 (CH₂), 107.4 (C-4), 116.3 (C-3a), 118.4 (C-2′, C-6′), 119.9 (C-7a), 121.3 (C-4′), 128.7 (C-3′, C-5′), 135.3 (C-5), 140.8 (C-3), 141.7 (C-7), 157.9 (C-1′), 168.7 (CO). HR-MS (ESI) m/z: Calcd for C₁₆H₁₉N₆O: [M1+H]⁺=311.1615. Found 311.1617. Anal. Calcd for C₁₆H₁₈N₆O: C, 61.92; H, 5.85; N, 27.08. Found: C, 61.68; H, 5.71; N, 27.37.
• Data for 39b: Yield 95%. Beige foam. ¹H-NMR (400 MHz, DMSO-d₆) δ: 2.31 (3H, s, CH₃), 2.52–2.70 (8H, br s, piperazine H-2″, H-3″, H-5″, H-6″), 3.25 (2H, s, CH₂), 6.97 (1H, t, H-4′, J=7.3 Hz), 7.12 (1H, d, H-4, J=5.8 Hz), 7.33 (2H, t, H-3′, H-5′, J=7.8 Hz), 7.69 (1H, d, H-5, J=5.8 Hz), 7.93 (2H, d, H-2′, H-6′, J=7.8 Hz), 9.13 (1H, br s, D₂O exchang., NH–aniline), 10.20 (1H, br s, D₂O exchang., NHCO), 13.11 (1H, br s, D₂O exchang., NH–pyrazole). ¹³C-NMR (50 MHz, DMSO-d₆) δ: 45.0 (CH₃), 51.9 (piperazine C-2″, C-6″), 54.2 (piperazine C-3″, C-5″), 60.5 (CH₂), 107.4 (C-4), 116.3 (C-3a), 118.4 (C-2′, C-6′), 119.7 (C-7a), 121.3 (C-4′), 128.7 (C-3′, C-5′), 135.3 (C-5), 140.9 (C-3), 141.9 (C-7), 158.1 (C-1′), 168.2 (CO). HR-MS (ESI) m/z: Calcd for C₁₉H₂₄N₇O: [M1+H]⁺=366.2037. Found 366.2044. Anal. Calcd for C₁₉H₂₃N₇O: C, 62.45; H, 6.34; N, 26.83. Found: C, 62.71; H, 6.48; N, 26.59.

Theoretical Calculations

The crystal structure of human GSK3β (pdb id: 1UV5) was utilized for docking calculations using the modules of Schrodinger Small-Molecule Drug Discovery Suite 2016.^21,26) Protein preparation was performed by the corresponding routine as implemented in Maestro. Prior to calculations, the studied compounds were prepared in terms of correct protonation states, tautomerism and stereoisomerism using the LigPrep routine of Maestro. Rigid docking was performed using the Glide-SP algorithm. The Van der Waals atom radii scaling was set to 0.8 for both the protein and docked ligands. The best poses were redocked by utilizing a flexible representation of the active site residues in a 5 Å sphere around the ligand using MacroModel. The OPLS2005 force-field, an implicit GB/SA water solvent model and the TNCG minimization algorithm were used.

Kinase Activity Evaluation

Kinase activities were assayed for each protein kinase in the appropriate buffers (listed below), with either a protein or a peptide as substrate in the presence of 15 µM [γ-³³P] ATP (3000 Ci/mmol; 10 mCi/mL) in a final volume of 30 µL following an assay previously described.²⁷⁾ Controls were performed with appropriate dilutions of dimethylsulfoxide. Full-length kinases are used unless specified. Peptide substrates were obtained from Proteogenix (Oberhausbergen, France).

• Buffers:
  • (A) 10 mM MgCl₂, 1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM dithiothreitol (DTT), 25 mM Tris–HCl pH 7.5, 50 µg/mL heparin, 0.15 mg/mL of BSA +0.23 mg/mL of DTT
  • (B) 60 mM β-glycerophosphate, 30 mM p-nitrophenylphosphate, 25 mM MOPS (pH 7), 5 mM EGTA, 15 mM MgCl₂, 1 mM DTT, 0.1 mM sodium orthovanadate
  • (D) 25 mM MOPS, pH 7.2, 12.5 mM β-glycerolphosphate, 25 mM MgCl₂, 5 mM EGTA, 2 mM ethylenediaminetetraacetic acid (EDTA), 0.25 mM DTT
  • (H) MOPS 25 mM pH 7.5, 10 mM MgCl₂
  • (K) Tris 50 mM pH 7.5, 20 mM MgCl₂, 2 mM MnCl₂
  • (R) 5 mM MOPS pH 7.2, 2.5 mM β-glycerophosphate, 4 mM MgCl₂, 2.5 mM MnCl₂, 1 mM EGTA, 0.4 mM EDTA, 50 µg/mL BSA, 0.05 mM DTT

• Each protein kinase was tested as follow:
  • HsRIPK3 (human, recombinant, expressed in Sf9 insect cells infected with 6xHIS : HsRIPK3 specific baculoviruses) was assayed in buffer R with 0.1 µg/µL of MBP (Myelin Basic Protein) as substrate.
  • HsHaspin-kd (human, kinase domain, amino acids 470 to 798, recombinant, expressed in bacteria) was assayed in buffer H with 0.007 µg/µL of Histone H3 (1–21) peptide (ARTKQTARKSTGGKAPRKQLA) as substrate.
  • HsCDK5/p25 (human, recombinant, expressed in bacteria) was assayed in buffer B, with 0.8 µg/µL of histone H1 as substrate.
  • HsAuroraB (human, recombinant, expressed by baculovirus in Sf9 insect cells, SignalChem, product #A31-10G) was assayed in buffer D with 0.2 µg/µL of MBP as substrate.
  • SscGSK3α/β (glycogen synthase kinase-3, porcine brain, native, affinity purified) was assayed in buffer A with 0.010 µg/µL of GS-1 peptide, a GSK-3-selective substrate (YRRAAVPPSPSLSRHSSPHQSpEDEEE, “Sp” stands for phosphorylated serine).
  • SscCK1δ/ε (casein kinase 1δ/ε, porcine brain, native, affinity purified) was assayed in buffer B, with 0.022 µg/µL of the following peptide: RRKHAAIGSpAYSITA as CK1-specific substrate.
  • RnDYRK1A-kd (Rattus norvegicus, amino acids 1 to 499 including the kinase domain, recombinant, expressed in bacteria, DNA vector kindly provided by Dr. W. Becker, Aachen, Germany) was assayed in buffer A with 0.033 µg/µL of the following peptide: KKISGRLSPIMTEQ as substrate.
  • MmCLK1 (from Mus musculus, recombinant, expressed in bacteria) was assayed in buffer A with 0.027 µg/µL of the following peptide: GRSRSRSRSRSR.

Acknowledgments

We thank the Cancéropôle Grand Ouest (axis: natural sea products in cancer treatment), IBiSA (French Infrastructures en sciences du vivant: biologie, santé et agronomie) and Biogenouest (Western France life science and environment core facility network) for supporting KISSf screening facility. The authors are grateful to Dr. Orestis Argyros and Dr. Constantin Tamvakopoulos from the Biomedical Research Foundation Academy of Athens (BRFAA), Division of Pharmacology-Pharmacotechnology, for performing the cytotoxicity testing.

Conflict of Interest

The authors declare no conflict of interest.

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