Introduction

Among the various important parent ring systems of pyrazolopyridine, substituted 1H-pyrazolo[3,4-b]pyridine is known to have several biological activities such as anxyolitic, anticonvulsant, anti-inflammatory, analgesic, hypoglycemic, antipyretic, vasodilators, and antileishmanial (Hohn et al., 1972; Lynch et al., 1988; Bernardino et al., 1996a; Ahluwalia and Goyal, 1996; Quiroga et al., 1998; Bernardino et al., 1999; Mello et al., 2004). Recently our research group described the synthesis and antiviral activity of derivatives of the 1H-pyrazolo[3,4-b]pyridine and thieno[2,3-b]pyridine systems with promising results (Bernardino et al., 1996b; Azevedo et al., 2002a; Pinheiro et al., 2004; Bernardino et al., 2007).

In a attempt to explore the antiviral potential of 1H-pyrazolo[3,4-b]pyridine derivatives, we have studied their effects of these compounds on Herpes simplex virus type 1 (HSV-1), Mayaro virus (MAY), and vesicular stomatitis virus (VSV). Herpes simplex type 1 (HSV-1) infects mucocutaneous epithelial cells and establishes latency in sensory ganglions (Whitley and Roizman, 2001). HSV-1 causes gingivostomatitis, cold sores, keratoconjunctivitis, and encephalitis. Although any viral protein essential for viral replication is a potential target, nearly all currently available drugs for herpes viruses are primarily inhibitors of viral DNA polymerase (Eizuru, 2003). Among the anti-herpes-virus agents, acyclovir, valaciclovir, penciclovir, famciclovir, idoxuridine, and trifluridine (applied topically) as well as brivudin are used in the treatment of herpes simplex virus (De Clercq, 2005).

Mayaro virus is a member of the Alphavirus genus, Togaviridae family and is antigenically closely related to Semiliki Forest virus (Casals and Whitman, 1957). Mayaro virus is an arbovirus, isolated for the first time in Trinidad, in 1954 and has since been reported as the cause of several epidemic outbreaks in Brazil, Bolivia, and other regions, mainly at colonial borders in the Amazon region (Causey and Maroja, 1957). Clinical manifestations of human infection have been described as feverish illness accompanied by headache, chills, nausea, photophobia, myalgia, and arthralgia. In some cases there is true arthritis that persists for months (Strauss and Strauss, 1994). Vesicular stomatitis virus (VSV) is a single-strand RNA virus of the Rhabdoviridae family. VSV has been isolated from a variety of animals and causes nonfatal disease of significant economic importance in cattle and swine (Rose and Whitt, 2007).

In order to identify the 1H-pyrazolo[3,4-b]pyridine system as promising compounds for the development of new antiviral agents, we synthesized a new set of derivatives 4-(phenylamino)/4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridines (2–5). The aim of this study was to assess their cytotoxicity and the potential antiviral activity.

Results and discussion

Chemistry

The synthesis of new 4-(phenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid (3a–l) derivatives and the new 4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid (5a–c) derivatives was achieved with an efficient synthetic route outlined in Scheme 1. A halogen in the C-4 position and an ester group in the C-5 position of 1H-pyrazolo[3,4-b]pyridine react readily with nucleophilic compounds to form a good precursor for the synthesis of these derivatives. Ethyl 4-chloro-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (1) was available in our laboratory and could be easily prepared from 5-aminopyrazoles through condensation with diethyl ethoxymethylenemalonate followed by chlorocyclization with phosphorus oxychloride (Hohn et al., 1971; Azevedo et al., 2002a; Azevedo et al., 2002b). Ethyl 4-chloro-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (1) on fusion with appropriate substituted anilines or aminopicolines gave the required new ethyl 4-(phenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylates (2a–l) (52–82%) or new ethyl 4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylates (4a–c) (50–60%), respectively. Subsequent hydrolysis of the ester afforded the corresponding 4-(phenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acids (3a–l) in high yields (86–93%) and 4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acids (5a–c) in high yields (80–93%) (Azevedo et al., 2002b).

Scheme 1
scheme 1

2a, 3a R = H, 2b, 3b R = p-CH3, 2c, 3c R = m-Cl, 2d, 3d R = m-NO2, 2e, 3e R = p-NO2, 2f, 3f R = m-Br, 2g, 3g R = p-Br, 2h, 3h R = m-CH3, 2i, 3i R = p-Cl, 2f, 3f R = m-OCH3, 2g, 3g R = p-OCH3

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The structures of esters and acids were determined by infrared (IR), 1H and 13C nuclear magnetic resonance (NMR), and mass spectroscopy. In the IR spectra, the carbonyl group absorptions were observed in the acids and esters derivatives at 1649–1678 cm−1 and 1672–1696 cm−1, respectively. In the 3435–3125 cm−1 region, NH bands were observed. The OH bands for the acid compounds were observed in the 3491–2500 cm−1 region. 1H NMR spectra indicated a chemical shift of the NH in the range of δ = 10.98–9.46 ppm as singlet signals. The aromatic protons signals in aniline and aminopicoline moieties appeared as a multiplet in orto derivatives or a double doublet in para derivatives. The quartet and triplet signals of the esters derivatives (2a–l and 4 a-c) appeared in the ranges δ = 4.62–4.42 ppm and 1.58–1.50 ppm, respectively. The singlet signals of the acids (3a–l and 5a–c) appeared in the range δ = 13.00–11.36 ppm.

Biological evaluation

Initially, the anti-herpetic effect of all compounds investigate was evaluated in a primary screening using the 50% end-point titration method described by Reed and Muench (1938).

As shown in Table 1, compounds 2a, 2d, 2h, 2i, 2j, 4b, and 4c exhibited the highest anti-HSV-1 activity, with the esters being, in general, more effective inhibitors than the corresponding acids. In conclusion, the introduction of ester group at C-5 in the 1H-pyrazolo[3,4-b]pyridine system increased activity against HSV-1 virus.

Table 1 Anti-HSV-1 virus activity of 1H-pyrazolo[3,4-b]pyridine derivatives 2a– l, 3a– l, 4a–c , and 5a– c
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To calculate the selective or therapeutic index of each compound for antiviral activity we infected Vero cells with HSV-1, Mayaro, and VSV to determine the EC50 value by plaque-reducing assay. The cytotoxicity of the compounds was determined in parallel by the MTT 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide colorimetric method (Mossmann, 1983) (Table 2). As shown in the Table 3, the effects of the compounds 3a and 3c, selected on the basis of preliminary screening (data not shown), exhibit the best EC50 values against VSV and Mayaro virus replication cycle.

Table 2 Anti-HSV-1 virus activity, cytotoxicity, and selectivity index in Vero cells for 1H-pyrazolo[3,4-b]pyridine derivatives
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Table 3 Anti-Mayaro and VSV virus activity, cytotoxicity, and selectivity index in Vero cells for 1H-pyrazolo[3,4-b]pyridine derivatives
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Conclusion

In summary, 15 new derivatives of the 1H-pyrazolo[3,4-b]pyridine system 2a–l, 3a–l, 4a–c, and 5a–c with different substituents were synthesized and exhibited a range of significant anti-herpes, anti-Mayaro, and anti-VSV activities, suggesting that these compounds are potential antivirus agents. Compounds 2d, 3f, 3a, and 3c exhibited antiviral activity against HSV-1, MAY, and VSV virus, with EC50 values of 6.8, 2.2, 4.8, 0.52, 2.5, and 1.0. None of the compounds showed toxicity for Vero cells. The mechanism of antiviral activity is unknown and requires further study, which is in progress in our laboratory.

Experimental section

1H and 13C NMR spectra were obtained at 300 MHz and 75 MHz, respectively, using a Varian Unity Plus instrument with tetramethylsilane as an internal standard. The chemical shifts (δ) are reported in ppm and the coupling constants (J) in Hertz. Fourier-transform infrared (FT-IR) spectra were recorded in a Perkin–Elmer Spectrum One instrument. The solid samples were determined in potassium bromide (KBr) pellets. Melting points (m.p.) were determined with a Fisher–Johns apparatus. Thin-layer chromatography (TLC) was carried out using silica gel F-254 glass plate (20 × 20 cm). All reagents and solvents used were analytical grade. The electron-ionization mass spectrometry (EI-MS) spectra were recorded using a Finingan MAT 711 A instrument. The ionization energy was 70 eV with a source temperature of 200°C and an accelerative voltage of 8 kV. Samples were introduced by using a standard direct-insertion probe. High-resolution data were obtained with the instrument using 10000 resolution).

Ethyl 4-chloro-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (1)

The procedure developed for the synthesis of 1 started from the previously prepared 5-amino-1-phenyl-1H-pyrazole. The condensation of aminopyrazole with diethyl ethoxymethylenemalonate, based on the Gould–Jacobs method, produced ethyl α-carboetoxi-β-(N-5-pyrazolylamino)acrylate, which was recrystallized from anhydrous ethanol. The resulting acrylate was mixed with phosphorus oxychloride and refluxed for 5 hours. The excess solvent was removed under reduced pressure and the resulting material was poured onto crushed ice and the product collected by filtration. The solid material was recrystallized from ethanol to yield ethyl 4-chloro-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (1) (Azevedo et al., 2002b).

General procedure for the preparation of the ethyl 4-(arylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylates (2a–l)

An equimolar mixture of 1 (1,2 g, 4 mmol) and a slight excess of the appropriate aniline (5 mmol), in 10 ml N,N-dimethylformamide (DMF) was heated in a silicone oil bath under reflux for 4 hours. The reaction mixture, after cooling, was poured into 50 ml of ice-cold water. The precipitate was filtered, dried, and recrystallized from a mixture of ethanol and water. The compounds were isolated in good yields (52–82%). The structures of the compounds were elucidated by IR, 1H and 13C NMR, and mass spectrometry.

2a. Ethyl 4-(phenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate

Yield: 1.08g (76%), m.p.: 197°C; IR (KBr, cm−1): (ν NH 3420; ν C = O 1678); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 6,81(s, H-3); 9,04(s, H-6); 8,08(d, 8.7, H-2′,6′), 7,51-7,26(10H, m), 4,42(q, 7.1, CH2), 1,42(t, 7.1, CH3), 10,61(s, N-H);13C NMR (DMSO-d6, δ in ppm) 135.7(C-3), 99.7(C-3a), 137.4(C-4), 105.1(C-5), 149.5(C-6), 150.3(C-7a), 131.9(C-1′), 121.5(C-2′, 6′), 128.3(C-3′, 5′), 126.8(C-4′), 158.1(C-1″), 120.4(C-2″), 128.5(C-3″), 140.1(C-4″), 128.5(C-5″), 120.4(C-6″), 164.9(CO2Et), 60.5(CH2), 13.7(CH3);

2b. Ethyl 4-(4′-methylphenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate

Yield: 0.93 g (63%), m.p.: 155°C; IR (KBr, cm-1): (ν NH 3435; ν C = O 1678); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 6.73(s, H-3), 9.04(s, H-6), 8.23(d, 8.7, H-2′,6′), 7.67(dd, 7.5, H-3′,5′), 7.54–7.46(5H, m), 4.53(q, 7.2, CH2), 1.53(t, 7.2, CH3), 10.52(s, N-H), 3.38(s, Ar-CH3); 13C NMR (DMSO-d6, δ in ppm) 135.5(C-3), 101.7(C-3a), 139.6(C-4), 105.4(C-5), 153.6(C-6), 151.7(C-7a), 137.3(C-1′), 122.4(C-2′, 6′), 131.3(C-3′, 5′), 130.0(C-4′), 158.6(C-1″), 122.4(C-2″), 128.1(C-3″), 138.6(C-4″), 128.1(C-5″), 122.4(C-6″), 168.6(CO2Et), 21.7(Ar-CH3), 61.6(CH2), 15.1(CH3).

2c. Ethyl 4-(3′-chlorophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate

Yield: 1.06 g (68%), m.p.: 182°C; IR (KBr, cm−1): (ν NH 3208; ν C = O 1681); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 6.92(s, H-3), 9.03(s, H-6), 8.20(d, 8.7, H-2′,6′), 7.68–7.56(5H, m), 7.47(dd, 7.5, H-4′), 7.73(dd, 8.7, H-5″), 4.50(q, 7.2, CH2), 1.47(t, 7.2, CH3),10.57(s, N-H); 13C NMR (DMSO-d6, δ in ppm) 133.9(C-3), 101.(C-3a), 138.3(C-4), 104.4(C-5), 152.2(C-6), 152.0(C-7a), 133.5(C-1′), 121.1(C-2′, 6′), 130.4(3′, 5′), 128.6(C-4′), 158.8(C-1″), 124.8(C-2″), 149.1(C-3″), 140.4(C-4″), 127.0(C-5″), 126.1(C-6″), 167.0(CO2Et), 60.3(CH2), 13.7(CH3); EI (70 eV) m/z (%): M+. 392.10435 (95), 346.06091 (100), 311.09595 (25), 283.09918 (15), 173.03200 (7), 110.99878 (4), 77.03763 (10)

2d. Ethyl 4-(3′-nitrophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate

Yield: 0.96 g (60%), m.p.: 173°C; IR (KBr, cm-1): (ν NH 3207; ν C = O 1681); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 7.25(s, H-3), 9.13(s, H-6), 8.30(d, 8.7, H-2′,6′), 7.74(dd, 7.5, H-3′,5′), 7.55(dd, 7.5, H-4′), 8.50(s, H-2″), 8.44(d, 8.1, H-4″), 7.97(dd, 8.7, H-5″), 8.10(d, 8.7, H-6″), 4.55(q, 7.2, CH2), 1.55(t, 7.2, CH3), 10.75(s, N-H); 13C NMR (DMSO-d6, δ in ppm) 134.8(C-3), 102.7(C-3a), 138.8(C-4), 105.2(C-5), 153.0(C-6), 152.6(C-7a), 133.2(C-1′), 122.1(C-2′, 6′), 129.4(C-3′, 5′), 126.9(C-4′), 158.9(C-1″), 120.9(C-2″), 148.9(C-3″), 140.9(C-4″), 121.9(C-5″), 120.9(C-6″), 167.6(CO2Et), 61.2(CH2), 14.5(CH3); EI (70 eV) m/z (%): M+. 403.12863 (100), 357.09045 (70), 311.09466 (30), 283.10117 (30), 256.08533 (7), 77.03626 (11).

2e. Ethyl 4-(4′-nitrophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate

Yield: 1.11 g (69%), m.p.: 176°C; IR (KBr, cm-1): (ν NH 3208; ν C = O 1681); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 7.18(s, H-6), 9.09(s, H-6), 8.25(d, 8.7, H-2′,6′), 7.68(dd, 7.5, H-3′,5′), 7.50(dd, 7.5, H-4′), 7.74(d, 8.7, H-2″), 8.43(d, 8.7, H-3″), 8.43(d, 8.7, H-5″), 7.74(d, 8.7, H-6″), 4.45(q, 7.2, CH2), 1.46(t, 7.2, CH3), 10.67(s, N-H); 13C NMR (DMSO-d6, δ in ppm) 134.4(C-3), 104.1(C-3a), 138.3(C-4), 105.6(C-5), 152.1(C-6), 152.0(C-7a), 133.6(C-1′), 121.1(C-2′, 6′), 128.8(C-3′, 5′), 126.3(C-4′), 158.7(C-1″), 123.3(C-2″), 124.9(C-3″), 144.0(C-4″), 124.9(C-5″), 124.9(C-6″), 166.7(CO2Et), 60.7(CH2), 13.8(CH3); EI (70 eV) m/z (%): M+. 403.12461 (100), 357.07820 (45), 311.08910 (80), 283.08977 (30), 256.08447 (7), 77.03500 (11).

2f. Ethyl 4-(3′-bromophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate

Yield: 1.31 g (75%), m.p.: 180°C; IR (KBr, cm−1): (ν NH 3224; ν C = O 1679); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 6.88(s, H-3), 8.99(s, H-6), 8.17(d, 8.7, H-2′,6′), 7.64–7.57(3H, m), 7.43(dd, 7.5, H-4′), 7.82(s, H-2″), 7.75(dd, 7.5, H-5″), 7.75(d, 7.5, H-6″), 4.44(q, 7.5, CH2), 1.44(t, 7.5, CH3),10.53(s, N-H); 13C NMR (DMSO-d6, δ in ppm) 134.7(C-3), 102.0(C-3a), 138.9(C-4), 104.9(C-5), 153.0(C-6), 153.2(C-7a), 133.0(C-1′), 121.8(C-2′, 6′), 129.1(C-3′, 5′), 126.8(C-4′), 158.0(C-1″″), 120.0(C-2″), 150.1(C-3″), 139.0(C-4″), 121.0(C-5″), 120.0(C-6″), 167.8(CO2Et), 61.0(CH2), 14.4(CH3); EI (70 eV) m/z (%): M+. 436.05399 (85), 392.00998 (55), 358.13943 (10), 311.09562 (100), 283.09590 (30), 256.08710 (8), 222.57337 (15), 155.54822 (18), 95.07887 (23).

2g. Ethyl 4-(4′-bromophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate

Yield: 1.31 g (75%), m.p.: 187°C; IR (KBr, cm-1): (ν NH 3219; ν C = O 1683); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 6.88(s, H-3), 8.99(s, H-6), 8.15(d, 8.7, H-2′,6′), 7.60–7.50(7H, m), 4.45(q, 7.5, CH2), 1.40(t, 7.5, CH3), 10.50(s, N-H); 13C NMR (DMSO-d6, δ in ppm) 134.1(C-3), 101.4(C-3a), 138.4(C-4), 104.5(C-5), 153.3(C-6), 152.1(C-7a), 132.6(C-1′), 121.4(C-2′, 6′), 129.0(C-3′, 5′), 128.8(C-4′), 158.2(C-1″), 120.1(C-2″), 126.4(C-3″), 138.5(C-4″), 126.4(C-5″), 120.1(C-6″), 167.4(CO2Et), 60.5(CH2), 13.5(CH3); EI (70 eV) m/z (%): M+. 436.05358 (65), 390.01168 (15), 311.09221 (100), 283.09354 (20), 256.08226 (4), 155.54525 (12), 77.17351(10).

2h. Ethyl 4-(3′-methylphenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate

Yield: 0.99 g (67%), m.p.: 187°C; IR (KBr, cm−1): (ν NH 3210; ν C = O 1680); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 6.75(s, H-3), 9.03(s, H-6), 8.21(d, 8.7, H-2′,6′), 7.68–7.41(7H, m), 4.51(q, 7.2, CH2), 1.51(t, 7.2, CH3), 10.53(s, N-H), 3.37(s, Ar-CH3); 13C NMR (DMSO-d6, δ in ppm) 135.5(C-3), 101.1(C-3a), 138.4(C-4), 105.1(C-5), 153.0(C-6), 152.9(C-7a), 133.0(C-1′), 122.0(C-2′, 6′), 128.7(C-3′, 5′), 126.1(C-4′), 158.1(C-1″), 120.5(C-2″), 129.0(C-3″), 138.5(C-4″), 124.0(C-5″), 120.5(C-6″), 166.9(CO2Et), 1.7(Ar-CH3), 61.6(CH2), 15.1(CH3).

2i. Ethyl 4-(4′-chlorophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate

Yield: 1.00 g (64%), m.p.: 159°C; IR (KBr, cm−1): (ν NH 3218; ν C = O 1681); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 6.94(s, H-3), 9.05(s, H-6), 8.08(d, 8.7, H-2′,6′), 7.51–7.25(7H, m), 4.42(q, 7.1, CH2), 1.42(t, 7.1, CH3), 10.51(s, N-H); 13C NMR (DMSO-d6, δ in ppm) 134.4(C-3), 104.1(C-3a), 138.3(C-4), 105.6(C-5), 152.3(C-6), 152.6(C-7a), 133.0(C-1′), 121.2(C-2′, 6′), 128.8(3′, 5′), 126.3(C-4′), 158.4(C-1″), 123.3(C-2″), 126.6(C-3″), 145.7(C-4″), 126.6(C-5″), 123.3(C-6″), 166.7(CO2Et), 60.7(CH2), 13.8(CH3); EI (70 eV) m/z (%): M+. 392.10854 (95), 346.06119 (100), 311.08943 (25), 283.08789 (15), 173.03286 (7), 110.99432 (4), 77.03839 (10)

2j. Ethyl 4-(3′-methoxyphenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate

Yield: 0.97 g (63%), m.p.: 135°C; IR (KBr, cm−1): (ν NH 3435; ν C = O 1678); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 6.77(s, H-3), 8.95(s, H-6), 8.16(d, 8.7, H-2′,6′), 7.13(dd, 7.5, H-3′,5′), 7.41(dd, 7.5, H-4′), 7.51(s, H-2″), 7.55(d, 7.8, H-4″), 7.60(dd, 7.5, H-5″), 7.53(d, 7.8, H-6″),4.43(q, 7.0, CH2), 1.43(t, 7.0, CH3), 10.51(s, N-H), 3.86(s, Ar-OCH3); 13C NMR (DMSO-d6, δ in ppm) 135.8(C-3), 98.1(C-3a), 137.3(C-4), 101.6(C-5), 147.4(C-6), 149.9(C-7a), 131.8(C-1′), 118.6(C-2′, 6′), 127.8(C-3′, 5′), 126.3(C-4′), 157.6(C-1″), 120.2(C-2″), 148.5(C-3″), 109.8(C-4″), 123.5(C-5″), 110.9(C-6″), 164.6(CO2Et), 57.9(Ar-OCH3), 69.4(CH2), 11.4(CH3); EI (70 eV) m/z (%): M+. 388.15321 (100), 342.11180 (100), 271.10042 (5), 244.08777(2), 171.06055 (7), 77.04092 (6).

2k. Ethyl 4-(4′-methoxyphenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate

Yield: 0.80 g (52%), m.p.: 148°C; IR (KBr, cm−1): (ν NH 3435; ν C = O 1672); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 6.60(s, H-3), 8.98(s, H-6), 8.19(d, 8.7, H-2′,6′), 7.63(dd, 7.5, H-3′,5′), 7.45(dd, 7.5, H-4′), 7.22(d, 8.7, H-2″), 7.50(d, 8.7, H-3″), 7.22 (d, 8.7, H-5″), 7.22(d, 8.7, H-6″), 4.49(q, 7.2, CH2), 1.48(t, 7.2, CH3), 10.46(s, N-H), 3.96(s, Ar-OCH3); 13C NMR (DMSO-d6, δ in ppm) 134.0(C-3), 99.9(C-3a), 138.1(C-4), 103.9(C-5), 152.2(C-6), 150.8(C-7a), 131.0(C-1′), 120.9(C-2′, 6′), 128.6(C-3′, 5′), 125.9(C-4′), 158.5(C-1″), 114.6(C-2″), 128.5(C-3″), 151.8(C-4″), 128.5(C-5″), 114.6(C-6″), 167.1(CO2Et), 55.0(Ar-OCH3), 60.1(CH2), 13.7(CH3); EI (70 eV) m/z (%): M+. 372.15870 (85), 326.12064 (100), 300.13490 (9), 163.06130 (7), 77.04092 (4).

2l. Ethyl 4-(3′-fluorophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate

Yield: 1.23 g (82%), m.p.: 206°C; IR (KBr, cm−1): (ν NH 3249; ν C = O 1676); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 7.03(s, H-3), 9.13(s, H-6), 8.30(d, 8.7, H-2′,6′), 7.77–7.53(7H, m), 4.57(q, 7.5, CH2), 1.55(t, 7.0, CH3), 10.67(s, N-H); 13C NMR (DMSO-d6, δ in ppm) 134.2(C-3), 104.0(C-3a), 138.4(C-4), 105.7(C-5), 153.0(C-6), 152.1(C-7a), 135.0(C-1′), 121.0(C-2′, 6′), 128.0(C-3′, 5′), 127.0(C-4′), 158.7(C-1″), 123.2(C-2″), 148.0(C-3″), 142.1(C-4″), 120.3(C-5″), 112.6(C-6″″), 167.0(CO2Et), 60.5(CH2), 13.5(CH3); EI (70 eV) m/z (%): M+. 376.12275 (90), 330.08058 (100), 303.07368 (10), 236.20421 (7), 165.03244 (11), 83.07803 (14).

General procedure for the preparation of the 4-(arylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acids (3a–l)

A mixture of 3 mmol of (2a–g), 10 ml of 20% sodium hydroxide solution, and 10 ml of ethanol was heated under reflux for 1–3 hours. On cooling the mixture was acidified with diluted hydrochloric acid (1:3), and the precipitate was filtered and recrystallized from a mixture of DMF and water. The compounds were isolated in excellent yields (86–93%). The structures of the compounds were elucidated by IR, 1H and 13C NMR, and mass spectrometry.

3a. 4-Phenylamino-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid

Yield: 0.85 g (86%), m.p.: 229°C; IR (KBr, cm−1): (ν OH 3435−2598; ν C = O 1654); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 7.01(s, H-3), 9.02(s, H-6), 8.23(d, 8.1, H-2′,6′), 7.86−7.45(8H, m), 10.78(s, N-H); 13C NMR (DMSO-d6, δ in ppm) 135.4(C-3), 100.0(C-3a), 151.7(C-4), 105.5(C-5), 153.1(C-6), 153.5(C-7a), 114.2(C-1′), 122.2(C-2′, 6′), 131.5(C-3′, 5′), 129.1(C-4′), 140.2(C-1″), 126.4(C-2″), 139.5(C-3″), 126.9(C-4″), 139.5(C-5″), 126.4(C-6″), 170.2(CO2H).

3b. 4-(4′-Methylphenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid

Yield: 0.94 g (92%), m.p.: 258°C; IR (KBr, cm−1): (ν OH 3430−2603; ν C = O 1654); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 6.67(s, H-3), 8.95(s, H-6), 8.18(d, 7.5, H-2′,6′), 7.61−7.40(7H, m), 10.73(s, N-H), 2.47(s, Ar-CH3); 13C NMR (DMSO-d6, δ in ppm) 134.8(C-3), 100.1(C-3a), 151.3(C-4), 104.5(C-5), 153.5(C-6), 152.8(C-7a), 139.1(C-1′), 121.7(C-2′, 6′), 130.7(C-3′, 5′), 129.3(C-4′), 139.1(C-1″), 126.6(C-2″), 137.8(C-3″), 136.7(C-4″), 137.8(C-5″), 126.6(C-6″), 170.3(CO2H), 21.0(Ar-CH3); EI (70 eV) m/z (%): M+. 344.13367 (95), 326.12041 (100), 297.10439 (5), 270.11045 (3), 199.93153 (4), 73.36787 (4).

3c. 4-(3′-Chlorophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid

Yield: 1.01 g (93%), m.p.: 236°C; IR (KBr, cm−1): (ν OH 3488−2766; ν C = O 1649); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 7.01(s, H-3), 9.07(s, H-6), 8.27(d, 7.5, H-2′,6′), 7.74−7.62 (5H, m), 7.52(dd, 7.5, H-4′), 7.79(s, H-2″), 10.92(s, N-H); 13C NMR (DMSO-d6, δ in ppm) 132.9(C-3), 100.5(C-3a), 148.6(C-4), 103.2(C-5), 151.6(C-6), 150.8(C-7a), 120.1(C-1′), 125.2(C-2′, 6′), 130.0(C-3′, 5′), 127.7(C-4′), 139.3(C-1″), 124.0(C-2″), 137.2(C-3″), 126.1(C-4″), 132.3(C-5″), 120.1(C-6″), 168.4(CO2H); EI (70 eV) m/z (%): M+. 364.07926 (100), 311.10332 (20), 283.10468 (9), 236.22030 (3), 155.55160 (7), 77.17758 (20).

3d. 4-(3′-Nitrophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid

Yield: 1.01 g (90%), m.p.: 255 °C, IR (KBr, cm−1): (ν OH 3401−2603; ν C = O l674); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 7.12(s, H-3), 9.04(s, H-6), 8.22(d, 7.5, H-2′,6′), 7.65(dd, 7.5, H-3′,5′), 7.46(dd, 7.5, H-4′), 8.42(s, H-2″), 8.35(d, 8.7, H-4″), 7.88(d, 8.7, H-5″), 8.03(d, 8.7, H-6″), 10.99(s, N-H); 13C NMR (DMSO-d6, δ in ppm) 138.2(C-3), 102.1(C-3a), 148.8(C-4), 104.3(C-5), 152.7(C-6), 152.0(C-7a), 120.2(C-1′), 126.1(C-2′, 6′), 132.0(C-3′, 5′), 130.5(C-4′), 148.1(C-1″), 121.3(C-2″), 140.0(C-3″), 128.7(C-4″), 134.0(C-5″), 120.9(C-6″), 168.2(CO2H); EI (70 eV) m/z (%): M+. 375.09687 (100); 310.09034 (27); 211.20865 (8); 178.57242 (23); 145.62161 (9); 121.49006 (10); 77.17135 (9).

3e. 4-(4′-Nitrophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid

Yield: 1.04 g (93%), m.p.: 256°C; IR (KBr, cm−1): (ν OH 3409−2603; ν C = O 1678); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 7.16(s, H-3), 9.06(s, H-6), 8.23(d, 7.5, H-2′,6′), 7.65(dd, 7.5, H-3′,5′), 7.46(dd, 7.5, H-4′), 7.74(d, 8.7, H-2″), 8.40(d, 8.7, H-3″), 8.40(d, 8.7, H-5″), 7.74(d, 8.7, H-6″), 10.98(s, N-H); 13C NMR (DMSO-d6, δ in ppm) 139.5(C-3), 130.3(C-3a), 148.2(C-4), 104.9(C-5), 153.7(C-6), 153.1(C-7a), 120.3(C-1′), 126.0(C-2′, 6′), 133.2(C-3′, 5′), 129.9(C-4′), 148.1(C-1″), 121.3(C-2″), 139.4(C-3″), 127.3(C-4″), 131.7(C-5″), 120.3(C-6″), 170.4(CO2H); EI (70 eV) m/z (%): M+. 375.09575 (100), 311.09014 (45), 283.09583 (26), 204.83632 (23), 138.51953 (12), 116.02373 (20), 83.21144 (14).

3f. 4-(3′-Bromophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid

Yield: 1.10 g (90%), m.p.: 246°C; IR (KBr, cm−1): (ν OH 3490−2580; ν C = O 1650); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 6.65(s, H-3), 8.72(s, H-6), 7.93(d, 7.5, H-2′,6′), 7.50−7.31(5H, m), 7.17(dd, 7.5, H-4′), 7.57(s, H-2″), 12.90(s, CO2H), 10.57(s, N-H); 13C NMR (DMSO-d6, δ in ppm) 134.4(C-3), 102.0(C-3a), 150.0(C-4), 104.7(C-5), 153.3(C-6), 152.6(C-7a), 120.5(C-1′), 125.9(C-2′, 6′), 130.5(C-3′, 5′), 129.6(C-4′), 141.0(C-1″), 121.5(C-2″), 138.8(C-3″), 126.6(C-4″), 131.7(C-5″), 120.5(C-6″), 170.0(CO2H); EI (70 eV) m/z (%): M+. 410.00421 (100), 339.37886 (8), 311.07759 (100), 210.05296 (7), 76.04006 (5).

3g. 4-(4′-Bromophenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid

Yield: 1.10 g (90%), m.p.: 265°C; IR (KBr, cm−1): (ν OH 3431−2580; ν C = O 1651); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 6.87(s, H-3), 8.95(s, H-6), 8.16(d, 7.5, H-2′,6′), 7.59(dd, 7.5, H-3′,5′), 7.40(dd, 7.5, H-4′), 7.50(d, 8.7, H-2″), 7.78(d, 8.7, H-3″), 7.78(d, 8.7, H-5″), 7.50(d, 8.7, H-6″), 13.00(s, CO2H), 10.73(s, N-H); 13C NMR (DMSO-d6, δ in ppm) 132.5(C-3), 101.5(C-3a), 149.9(C-4), 104.5(C-5), 153.0(C-6), 152.2(C-7a), 115.0(C-1′), 120.0(C-2′, 6′), 128.7(C-3′, 5′), 126.3(C-4′), 138.4(C-1″), 120.0(C-2″), 134.2(C-3″), 121.2(C-4″), 128.9(C-5″), 115.0(C-6″), 169.7(CO2H); EI (70 eV) m/z (%): M+. 410.00732 (13), 386.21067 (4), 311.08624 (100), 283.04687 (13), 82.30021 (5).

3h. 4-(3′-Methylphenylamino)-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid

Yield: 0.92 g (90%), m.p.: 237°C; IR (KBr, cm-1): (ν OH 3482−2592; ν C = O 1650); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 6.71(s, H-3), 8.95(s, H-6), 8.16(d, 7.5, H-2′,6′), 7.60(dd, 7.5, H-3′,5′), 7.52(dd, 7.5, H-4′), 7.44−7.32(4H, m), 10.81(s, N-H), 2.21(s, Ar-CH3); 13C NMR (DMSO-d6, δ in ppm) 138.9(C-3), 101.4(C-3a), 151.1(C-4), 104.7(C-5), 153.3(C-6), 152.6(C-7a), 124.4(C-1′), 127.8(C-2′, 6′), 130.0(C-3′, 5′), 129.4(C-4′), 140.0(C-1″), 126.7(C-2″), 139.1(C-3″), 128.9(C-4″), 134.8(C-5″), 121.7(C-6″), 170.3(CO2H), 21.3(Ar-CH3); EI (70 eV) m/z (%): M+. 344.13178 (95), 326.11951 (100), 297.11572 (5), 270.10950 (3), 199.93871 (4), 73.35493 (4).

3i. 4-(4′-Chlorophenylamino)-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid

Yield: 0.97 g (89%), m.p.: 268°C; IR (KBr, cm−1): (ν OH 3430−2603; ν C = O 1654); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 6.87(s, H-3), 8.97(s, H-6), 8.17(d, 7.5, H-2′,6′), 7.61(dd, 7.5, H-3′,5′), 7.42(dd, 7.5, H-4′), 7.58(d, 8.7, H-2″), 7.67(d, 8.7, H-3″), 7.67(d, 8.7, H-5″), 7.58(d, 8.7, H-6″), 10.77(s, N-H); 13C NMR (DMSO-d6, δ in ppm) 131.7(C-3), 101.4(C-3a), 150.0(C-4), 104.3(C-5), 153.0(C-6), 152.2(C-7a), 121.2(C-1′), 128.5(C-2′, 6′), 128.9(C-3′, 5′), 128.9(C-4′), 138.4(C-1″), 126.3(C-2″), 134.2(C-3″), 137.9(C-4″), 129.6(C-5″), 121.2(C-6″), 169.6(CO2H); EI (70 eV) m/z (%): M+. 364.07828 (100), 311.10296 (20), 283.11744 (9), 236.22865 (3), 155.54781 (7), 77.17234 (20).

3j. 4-(3′-Methoxyphenylamino)-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid

Yield: 0.97 g (90%), m.p.: 237°C; IR (KBr, cm−1): (ν OH 3330−2590; ν C = O 1654); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 6.91(s, H-3), 9.05(s, H-6), 8.27(d, 7.5, H-2′,6′), 7.69(dd, 7.5, H-3′,5′), 7.51(dd, 7.5, H-4′), 7.66−7.61(4H, m), 10.88(s, N-H), 3.95(s, Ar-OCH3); 13C NMR (DMSO-d6, δ in ppm) 139.3(C-3), 101.8(C-3a), 151.1(C-4), 105.0(C-5), 153.7(C-6), 153.0(C-7a), 114.3(C-1′), 122.0(C-2′, 6′), 131.3(C-3′, 5′), 129.7(C-4′), 140.7(C-1″), 119.6(C-2″), 161.0(C-3″), 127.0(C-4″), 135.2(C-5″), 113.1(C-6″), 170.6(CO2H), 56.1(Ar-OCH3); EI (70 eV) m/z (%): M+. 360.12379 (100), 298.08825 (10), 230.57425 (4), 171.06160 (8), 77.17826 (13).

3k. 4-(4′-Methoxyphenylamino)-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid

Yield: 0.98 g (91%), m.p.: 261°C; IR (KBr, cm−1): (ν OH 3430−2588; ν C = O 1654); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 6.70(s, H-3), 9.03(s, H-6), 8.26(d, 7.5, H-2′,6′), 7.69(dd, 7.5, H-3′,5′), 7.51(dd, 7.5, H-4′), 7.28(d, 8.7, H-2″), 7.56(s, H-3″), 7.56(d, 8.7, H-5″″), 7.28(d, 8.7, H-6″), 10.77(s, N-H), 4.02(s, Ar-OCH3); 13C NMR (DMSO-d6, δ in ppm) 131.3(C-3), 100.5(C-3a), 151.3(C-4), 104.1(C-5), 152.9(C-6), 152.2(C-7a), 114.1(C-1′), 121.1(C-2′, 6′), 128.7(C-3′, 5′), 126.1(C-4′), 138.5(C-1″), 120.0(C-2″), 134.2(C-3″), 158.7(C-4″), 128.8(C-5″), 114.0(C-6″), 169.8(CO2H), 55.2(Ar-OCH3); EI (70 eV) m/z (%): M+. 360.12451 (100), 298.08978 (10), 230.58327 (4), 171.07122 (8), 77.17399 (13).

3l. 4-(3′-Fluorophenylamino)-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid

Yield: 0.97 g (93%), m.p.: 258°C; IR (KBr, cm−1): (ν OH 3412−2586; ν C = O 1675); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 7.04(s, H-3), 9.08(s, H-6), 8.28(d,7.5, H-2′,6′), 7.79−7.54(6H, m), 7.49(dd,7.5, H-4′), 10.98(s, N-H); 13C NMR (DMSO-d6, δ in ppm) 134.8(C-3), 102.3(C-3a), 150.4(C-4), 105.0(C-5), 153.6(C-6), 152.9(C-7a), 115.0(C-1′), 121.9(C-2′, 6′), 129.6(C-3′, 5′), 126.9(C-4′), 141.4(C-1″), 121.9(C-2″), 139.2(C-3″), 123.3(C-4″), 131.9(C-5″), 114.8(C-6″), 170.4(CO2H); EI (70 eV) m/z (%): M+. 348.10136 (100), 304.11069 (7), 274.07411 (4), 198.04128 (4), 165.04218 (10), 77.17166 (12).

General procedure for the preparation of the ethyl 4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylates (4a–c)

An equimolar mixture of 1 (1.08 g, 4 mmol) and a slightly excess of the appropriate aminopicoline (5 mmoles) in 10 ml DMF was heated in a silicone oil bath under reflux for 4 hours. The reaction mixture, after cooling, was poured into 50 ml of ice-cold water. The precipitate was filtered, dried, and recrystallized from a mixture of ethanol and water. The compounds were isolated in good yields (50–60%). The structures of the compounds were elucidated by IR, 1H and 13C NMR, and mass spectrometry.

4a. Ethyl 4-[(4′-methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate

Yield: 0.72 g (60%), m.p.: 180°C; IR (KBr, cm−1): (ν NH 3110; ν C = O 1696); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 8.12(d, 7.5, H-2′,6′), 7.52(dd, 7.5, H-3′,5′), 7.33(dd, 7.5, H-4′), 7.33(dd, 7.5, H-3″), 7.52(dd, 7.5, H-4″), 8.13(d, 7.5, H-6″), 4.38(q, 7.0, CH2), 1.40(t, 7.0, CH3), 9.56 (s, N-H), 3.30 (s, Ar-CH3); 13C NMR (DMSO-d6, δ in ppm) 136.7(C-3), 107.0(C-3a), 145.5(C-4), 108.0(C-5), 152.7(C-6), 152.5(C-7a), 134.0(C-1′), 121.7(C-2′, 6′), 129.6(C-3′, 5′), 126.5(C-4′), 157.1(C-2″), 121.6(C-3″), 139.4(C-4″), 119.4(C-5″), 152.0(C-6″), 167.1(CO2Et), 26.1(Ar-CH3), 60.9(CH2), 14.6(CH3); EI (70 eV) m/z (%): M+. 373.4102 (100)

4b. Ethyl 4-[(5′-methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate

Yield: 0.70 g (50%), m.p.: 193°C; IR (KBr, cm−1): (ν NH 3110; ν C = O 1695); 1H NMR (DMSO-d6, J in Hz, δ in ppm) RMN 8.90 (s, H-3), 9.26 (s, H-6), 8.38 (d, 7.5, H-2′,6′), 7.82(dd, 7.5, H-3′,5′), 7.63(dd, 7.5, H-4′), 8.38(d, 7.5, H-6″), 4.62(q, 7.0, CH2), 1.58(t, 7.0, CH3), 3.50(s, Ar-CH3); 13C NMR (DMSO-d6, δ in ppm) 135.1(C-3), 101.2(C-3a), 145.4(C-4), 103.4(C-5), 152.4(C-6), 152.3(C-7a), 139.0(C-1′), 122.2(C-2′, 6′), 130.0(C-3′, 5′), 127.8(C-4′), 157.5(C-2″), 117.8(C-3″), 139.1(C-4″), 120.3(C-5″), 152.1(C-6″), 164.2(CO2Et), 21.9(Ar-CH3), 62.5(CH2), 14.8(CH3); EI (70 eV) m/z (%): M+. 373.4139 (100)

4c. Ethyl 4-[(6′-methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate

Yield: 0.70 g (50%), m.p.: 165°C; IR (KBr, cm−1): (ν NH 3125; ν C = O 1693); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 9.40 (s, H-3), 9.50 (s, H-6), 9.10 (d, 7.5, H-2′,6′), 8.30−7.20(6H, m), 4.45(q, 7.0, CH2), 1.45(t, 7.0, CH3), 2.20 (s, Ar-CH3); 13C NMR (DMSO-d6, δ in ppm) 136.2(C-3), 104.0(C-3a), 145.9(C-4), 102.8(C-5), 152.1(C-6), 152.9(C-7a), 136.7(C-1′), 122.4(C-2′, 6′), 129.1(C-3′, 5′), 126.9(C-4′), 156.8(C-2″), 120.2(C-3″), 139.7(C-4″), 119.6(C-5″), 152.9(C-6″), 164.9(CO2Et), 25.2(Ar-CH3), 60.5(CH2), 14.1(CH3); EI (70 eV) m/z (%): M+. 373.4186 (100)

General procedure for the preparation of the 4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acids (5a–c)

A mixture of 3 mmol of (4a–c), 10 ml of 20% sodium hydroxide solution, and 10 ml of ethanol was heated under reflux for 1–3 hours. On cooling mixture was acidified with diluted hydrochloric acid (1:3), and the precipitated was filtered and recrystallized from a mixture of DMF and water. The compounds were isolated in excellent yields (80–93%). The structures of the compounds were elucidated by IR, 1H and 13C NMR, and mass spectrometry.

5a. 4-[(4′-Methylpyridin-2-yl)amino]-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid

Yield: 0.73 g (80%), m.p.: > 300°C; IR (KBr, cm−1): (ν OH 3491−2580; ν C = O 1674); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 8.42(s, H-3), 9.07(s, H-6), 8.22(d, 8.0, H-2′,6′), 7.69(dd, 7.5, H-3′,5′), 7.51(dd, 7.5, H-4′), 7.90(d, 7.5,H-3″), 7.42(d, 7.5, H-4″), 7.73(s, H-6″), 11.37(s, CO2H), 3.56(s, Ar-CH3); 13C NMR (DMSO-d6, δ in ppm) 135.8(C-3), 102.4(C-3a), 145.3(C-4), 106.1(C-5), 152.5(C-6), 151.1(C-7a), 137.0(C-1′), 121.9(C-2′, 6′), 129.9(C-3′, 5′), 126.8(C-4′), 157.3(C-2″), 118.6(C-3″), 139.7(C-4″), 119.8(C-5″), 152.6(C-6″), 21.7(Ar-CH3), 170.9(CO2H); EI (70 eV) m/z (%): M+. 345.3629 (100)

5b. 4-[(5′-Methylpyridin-2-yl)amino]-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid

Yield: 0.85 g (93%), m.p.: 225°C; IR (KBr, cm−1): (ν OH 3426−2600; ν C = O 1678); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 8.81(s, H-3), 9.08(s, H-6), 8.23(d, 8.0, H-2′,6′), 7.61(dd, 7.5, H-3′,5′), 7.44(dd, 7.5, H-4′), 7.95(s, H-3″), 7.70(d, 7.5, H-5″), 8.43(d, 7.5, H-6″), 11.36(s, CO2H), 9.46(s, N-H), 3.27(s, Ar-CH3); 13C NMR (DMSO-d6, δ in ppm) 135.6(C-3), 102.8(C-3a), 145.7(C-4), 104.2(C-5), 152.1(C-6), 152.5(C-7a), 138.5(C-1′), 121.6(C-2′, 6′), 129.2(C-3′, 5′), 127.2(C-4′), 157.8(C-2″), 117.6(C-3″), 139.0(C-4″), 119.6(C-5″), 152.2(C-6″), 21.2(Ar-CH3), 170.2(CO2H); EI (70 eV) m/z (%): M+. 345.3678 (100)

5c. 4-[(6′-Methylpyridin-2-yl)amino]-l-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid

Yield: 0.78 g (85%), m.p.: 252°C; IR (KBr, cm−1): (ν OH 3400−2500; ν C = O 1678); 1H NMR (DMSO-d6, J in Hz, δ in ppm) 8.86(s, H-3), 9.10(s, H-6), 8.25(d, 8.0, H-2′,6′), 7.65(dd, 7.5, H-3′,5′), 7.46(dd, 7.5, H-4′), 7.36(d, 7.5, H-4″), 7.77(dd, 7.5, H-5″), 8.35(d, 7.5, H-6″), 11.45(s, CO2H), 9.51(s, NH), 3.31(s, Ar-CH3); 13C NMR (DMSO-d6, δ in ppm) 135.4(C-3), 102.2(C-3a), 145.1(C-4), 103.7(C-5), 152.8(C-6), 152.0(C-7a), 138.9(C-1′), 121.0(C-2′, 6′), 129.8(C-3′, 5′), 127.6(C-4′), 157.1(C-2″), 116.8(C-3″), 139.7(C-4″), 119.2(C-5″), 152.8(C-6″), 21.7(Ar-CH3), 171.2(CO2H); EI (70 eV) m/z (%): M+. 345.3634 (100)

Cell culture and virus

Vero cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS; HyClone, Logan, Utah), 100 U/mL penicillin and 100 ug/mL streptomycin, at 37°C in 5% CO2.

Cytotoxicity assay

A monolayer of about 104 Vero cells in 96-multiwell plates was treated with various concentrations of the compounds for the 72 h. Then, 50 μl of a 1 mg/mL solution of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT; Sigma) was added to evaluate cell viability according to procedures described elsewhere (Mossmann, 1983). The 50% cytotoxic concentration (CC50) was calculated by linear regression analysis of the dose–response curves.

Plaque-reduction assay

The assay followed procedures described previously (Lucero et al., 2006). Acyclovir was used as a positive control. Vero cells in six-well plates were exposed to different dilutions of the supernatant from the assay described above for 1 h at 37°C. Residual viruses were then rinsed with PBS, and DMEM containing 5% FBS and 1% methylcellulose (Fluka) (overlay medium) was added to cells. After 72 h at 37°C, the monolayers were fixed with 10% formaldehyde in PBS and stained with a 0.1% solution of crystal violet in 70% methanol, and the virus titers were calculated by scoring the plaque-forming units (PFU). The 50% antiviral concentration (EC50) was calculated by linear regression analysis of dose–response curves.