The indazole scaffold is a pharmacophore with a wide spectrum of biological activity: anti-inflammatory, antimicrobial, antitumor, etc. [1–5]. Several natural compounds containing the indazole ring (nigellicine, nigeglanin, and nigellidine, Scheme 1) were isolated from Nigella glandulifera and Nigella sativa extracts [6–10]. Indazole derivatives are constituents of valuable pharmaceuticals. Some of them are bindarite, bendazac, benzydamine, lonidamine, and granisetron used as antitumor, anti-inflammatory, immunosuppressive, and serotonergic agents [11–17]. Indazole are promising agents in drug discovery. In this regard, development of synthetic ways for obtaining new compounds of indazole series is an urgent task.

Scheme
scheme 1

1.

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The reaction of 3-aryl-2,4-diacetyl-5-hydroxy-5-methylcyclocyclohexanones [18–20], alkyl 2-aryl-4hydroxy-4-methyl-6-oxocyclohexane-1,3-dicarboxylates [20–28] and N1,N3,2-triaryl-4-hydroxy-4-methyl-6-oxocyclohexane-1,3-dicarboxamides [29–30] with hydrazine hydrate has been known to afford tetrahydroindazoles, among which compounds with antimicrobial and anti-inflammatory activity have been found [24, 28, 30]. In order to search for new biologically active compounds, herein we studied the reaction of 2-aryl-4-hydroxy-4-methyl-6-oxocyclohexane-1,3-dicarboxamides 1а–1k with hydrazine hydrate. The reaction was performed upon boiling the reagents in ethyl alcohol in the absence of a catalyst. A series of new 4-aryl-3,6-dihydroxy-6-methyl-4,5,6,7-tetrahydro-2H-indazole-5-carboxamides 2а–2k was obtained in 28–83% yields (Scheme 2). Probably, the formation of compounds 2а–2k proceeds through the intermediate formation of hydrazone A (Scheme 1). The latter undergoes cyclization to form the indazole ring. Compounds 2a–2k are white, pale yellow or pink crystalline substances, soluble in dimethyl sulfoxide, dimethylformamide, when heated in ethyl and isopropyl alcohols, in acetone, insoluble in water. In the IR spectra of crystalline samples of compounds 2а–2k, there are stretching vibration bands in the ranges of 3470–3610 (OH), 3260–3452 (NH), and 1628–1674 cm–1 (CON). In the 1H NMR spectra of indazole derivatives 2а–2k, a singlet of the methyl group (1.24‒1.29 ppm), two doublets of CH group at positions 4 and 5 of the ring (2.29‒2.42 and 3.81‒4.13 ppm, J = 10‒10.5 Hz) are observed. Two doublet signals at 2.51‒2.61 and 2.57‒2.72 ppm (J = 16.0‒16.7 Hz) correspond to the protons of the methylene group at position 7 of the ring. A singlet in 4.55‒4.65 ppm range belongs to the hydroxyl group. In the weak field, two singlets of the amide group NH2 (6.92‒7.16 ppm) and a broadened signal of two protons of the pyrazole ring (9.6‒10.7 ppm) are recorded. The 13C NMR spectra of compounds 2а, 2b, 2d, 2f, 2h, and 2j show signals at 28.56‒28.88 (CH3), 157.80‒158.08 (=C3OH), 174.91‒176.09 ppm (C5ONH2). According to the published data [26, 30–31], indazoles 2a–2k can exist in the form of enol B, C or ketone D, E forms (Scheme 3). Compounds 2а–2k gives deep red color on reaction with neutral ferric chloride, which indicates the existence of the enol form in the solution and does not contradict the data on the structure of analogs, which was confirmed by single crystal X-ray diffraction analysis [31]. To determine the structure of 4,5,6,7-tetrahydro-2H-indazole-5-carboxamides 2а–2k in the crystalline state, a single crystal of compound 2а obtained by slow crystallization from an alcohol solution was analyzed by X-ray diffraction method. Compound 2а crystallizes as a solvate with ethanol in a 1 : 1 ratio in the monoclinic space group (Fig. 1, ethanol molecule is omitted). The bond lengths in the pyrazolone moiety [for example, C1=O1 1.243(3) Å] is more typical for the carbonyl rather than for the enol form. Intermolecular hydrogen bonds and the localization of hydrogen atoms H1 and H2 unambiguously indicate the existence of compound 2а in the crystal in the ketone form D. The average deviation of the pyrazole ring atoms from the root mean square plane is 0.032 Å. The cyclohexene ring is in the half-chair conformation: the C5 and C6 atoms are out off of the C4C3C2C7 plane by 0.35 and –0.37 Å, respectively. The crystal contains only one diastereomer of compound 2а with the relative configuration S*, R*, S* of atoms C5, C6, and C7, respectively. Hydroxyl and carbamoyl groups are cis-positioned relative to each other and form an intramolecular hydrogen bond O2–H2A∙∙∙O3. In the crystal of 2a solvate, intermolecular hydrogen bonds OH∙∙∙O and NH∙∙∙O form three-dimensional network.

Scheme
scheme 2

2.

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Scheme
scheme 3

3.

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Fig. 1.
figure 1

General view of the molecule of compound 2а in the crystal.

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Compounds 2а, 2b, 2d, 2e, 2g, and 2k were tested for antimicrobial activity against reference strains of Escherichia coli 25922 ATCC and Staphylococcus aureus 6538P ATCC. The minimum inhibitory concentrations (from 500 to 1000 μg/mL) indicate low antimicrobial activity.

In addition, compounds 2а, 2b, 2d, 2e, 2g, and 2k were tested for analgesic activity (Table 1). In the hot plate test, all the compounds exhibited analgesic activity, since the duration of the animals’ stay on the heated plate significantly increased compared to the control. Analgesic activity of the tested compounds did not differ from the reference drugs. The tested compounds (except for 2g) reduced the nociceptive response of the animals compared to the control group. The most pronounced decrease in the number of writhing (10.75±2.46) was found when using compound 2k (control 27.25±2.43). In conclusion, the reaction of substituted 6-oxocyclohexane-1,3-dicarboxamides with hydrazine hydrate in the absence of a catalyst yielded a series of new 4-aryl-3,6-dihydroxy-6-methyl-4,5,6,7-tetrahydro-2H-indazole-5-carboxamides exhibiting analgesic activity.

Table 1. Analgesic activity of 4-aryl-3,6-dihydroxy-6-methyl-4,5,6,7-tetrahydro-2H-indazole-5-carboxamidesa
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IR spectra were recorded in mineral oil on a FSM-1202 Fourier spectrometer. 1H and 13C NMR (DMSO-d6) spectra were recorded on a Bruker DRX 400 spectrometer with operating frequencies of 400 and 100 MHz, respectively. Elemental analysis was performed on a PerkinElmer 2400 apparatus. Melting points were determined on a Melting Point M-565 apparatus.

Single crystal X-ray diffraction analysis was performed on an Xcalibur Ruby diffractometer equipped with a CCD detector according to the standard procedure [MoKα-radiation, 295(2) K, ω-scanning with a step of 1°]. Absorption was taken into account empirically using the SCALE3 ABSPACK algorithm [32]. Crystal system (C15H17N3O3∙C2H6O, M 333.38) is monoclinic, space group P21/c, a 12.088(3), b 12.792(2), c 12.016(2) Å, β 109.98(2)°, V 1746.3(7) Å3, Z 4, dcalc 1.268 g/cm3, μ 0.091 mm–1. The structure was solved using the SHELXS program [33] and refined by full-matrix least squares in F2 in anisotropic approximation for all non-hydrogen atoms using the SHELXL software [34] with the OLEX2 graphical interface [35]. Hydrogen atoms were refined using the rider model (except for the hydrogen atoms of the NH, NH2, and OH groups, refined independently in the isotropic approximation). Final refinement parameters are: R1 0.0685 [for 2839 reflections with I > 2σ(I)], wR2 0.2013 (for all 4198 independent reflections), S 1.023. Crystallographic data were deposited in the Cambridge Crystallographic Data Center (CCDC 2032605) and can be requested at www.ccdc.cam.ac.uk/data_request/cif.

2-Aryl-4-hydroxy-4-methyl-6-oxocyclohexane-1,3-dicarboxamides 1а–1k were prepared according to a known procedure [36].

3,6-Dihydroxy-6-methyl-4-phenyl-4,5,6,7-tetrahydro-2H-indazole-5-carboxamide (2а). To a solution of 0.003 mol of 4-hydroxy-4-methyl-2-phenyl-6-oxocyclohexane-1,3-dicarboxamide 1a in 25 mL of ethyl alcohol was added dropwise a 20% excess of hydrazine hydrate. The reaction mixture was refluxed for 4 h, then cooled. The crystals were filtered off and recrystallized from ethyl alcohol. Yield 57%, mp 284–286°C (ethanol). IR spectrum, ν, cm–1: 3472 (OH), 3425, 3343 (NH), 1670 (CON). 1H NMR spectrum, δ, ppm: 1.27 s (3Н, CH3), 2.38 d (1Н, С4Н, J = 10.2 Hz), 2.57 d (1Н, С7НАНВ, J = 16.0 Hz), 2.63 d (1Н, С7НАНВ, J = 16.0 Hz), 3.97 d (1Н, С5Н, J = 10.2 Hz), 4.59 s (1Н, ОН), 6.95 s (1H, NH), 7.09‒7.23 м (5Н, С6Н5 + 1H, NH), 10.13 br. s (2Н, NH, OH). 13C NMR spectrum, δС, ppm: 28.77, 35.39, 39.24, 58.38, 70.16, 99.61, 125.76, 127.56, 127.56, 128.21, 138.73, 142.94, 158.03, 175.62. Found, %: C 62.65; H 5.92; N 14.62. C15H17N3O3. Calculated, %: C 62.71; H 5.96; N 14.63.

Compounds 2b–2k were obtained similarly.

3,6-Dihydroxy-6-methyl-4-(4-methylphenyl)-4,5,6,7-tetrahydro-2H-indazole-5-carboxamide (2b). Yield 45%, mp 286–288°C (ethanol). IR spectrum, ν, cm–1: 3592 (OH), 3404, 3303 (NH), 1674 (CON). 1H NMR spectrum, δ, ppm: 1.25 s (3Н, CH3), 2.26 s (3Н, 4-CH3С6Н4), 2.34 d (1Н, С4Н, J = 10.0 Hz), 2.54 d (1Н, С7НАНВ, J = 16.0 Hz), 2.61 d (1Н, С7НАНВ, J = 16.0 Hz), 3.91 d (1Н, С5Н, J = 10.2 Hz), 4.57 s (1Н, ОН), 6.92 s (1H, NH), 6.98 d (2Н, 4-CH3С6Н4, J = 8.0 Hz), 7.02 d (2Н, 4-CH3С6Н4, J = 8 Hz), 7.07 s (1H, NH), 10.00 br. s (2Н, NH, OH). 13C NMR spectrum, δС, ppm: 20.64, 28.79, 35.38, 38.82, 58.39, 70.16, 99.74, 128.09, 128.20, 134.45, 138.63, 139.88, 158.01, 175.71. Found, %: C 63.72; H 6.35; N 13.88. C16H19N3O3. Calculated, %: C 63.77; H 6.36; N 13.94.

3,6-Dihydroxy-4-(4-isopropylphenyl)-6-methyl-4,5,6,7-tetrahydro-2H-indazole-5-carboxamide (2c). Yield 35%, mp 252–254°С (ethanol). IR spectrum, ν, cm–1: 3610 (OH), 3338 (NH), 1670 (CON). 1H NMR spectrum, δ, ppm: 1.19 d [3Н, (CH3)2CH, J = 8.0 Hz], 1.21 d [3Н, (CH3)2CH, J = 8.0 Hz], 1.25 s (3Н, CH3), 2.34 d (1Н, С4Н, J = 10.0 Hz), 2.57 d (1Н, С7НАНВ, J = 16.4 Hz), 2.63 d (1Н, С7НАНВ, J = 16.4 Hz), 2.85 m [1Н, (CH3)2CH], 3.93 d (1Н, С5Н, J = 10.0 Hz), 4.55 s (1Н, ОН), 6.94 s (1H, NH), 7.02 d (2Н, C6H4, J = 8.0 Hz), 7.09 d (2H, C6H4, J = 8.0 Hz ), 7.11 s (1H, NH), 9.89 br. s (2Н, NH, OH). Found, %: C 65.58; H 7.11; N 12.71. C18H23N3O3. Calculated, %: C 65.63; H 7.04; N 12.76.

3,6-Dihydroxy-6-methyl-4-(3-nitrophenyl)-4,5,6,7-tetrahydro-2H-indazole-5-carboxamide (2d). Yield 57%, mp 284–286°C (ethanol). IR spectrum, ν, cm–1: 3482 (OH), 3380 (NH), 1668 (CON). 1H NMR spectrum, δ, ppm: 1.29 s (3Н, CH3), 2.40 d (1Н, С4Н, J = 10.5 Hz), 2.59 d (1Н, С7НАНВ, J = 16.0 Hz), 2.69 d (1Н, С7НАНВ, J = 16.0 Hz), 4.12 d (1Н, С5Н, J = 10.5 Hz), 4.65 s (1Н, ОН), 7.01 s (1H, NH), 7.15 s (1H, NH), 7.55 m (2Н, С6Н4), 7.92 s (1Н, С6Н4), 8.06 d (1Н, С6Н4, J = 8 Hz), 10.11 br. s (2Н, NH, OH). 13C NMR spectrum, δС, ppm: 28.56, 35.48, 39.11, 58.22, 70.11, 98.69, 121.12, 122.72, 129.12, 135.44, 139.11, 145.39, 147.57, 157.80, 174.91. Found, %: C 54.23; H 4.91; N 16.96. C15H16N4O5. Calculated, %: C 54.21; H 4.85; N 16.86.

3,6-Dihydroxy-4-[4-(dimethylamino)phenyl]-6-methyl-4,5,6,7-tetrahydro-2H-indazole-5-carboxamide (2e). Yield 46%, mp 282–284°C (ethanol). IR spectrum, ν, cm–1: 3470 (OH), 3434, 3335 (NH), 1656 (CON). 1H NMR spectrum, δ, ppm: 1.25 s (3Н, CH3), 2.32 d (1Н, С4Н, J = 10.3 Hz), 2.56 d (1Н, С7НАНВ, J = 16.0 Hz), 2.59 d (1Н, С7НАНВ, J = 16.0 Hz), 2.86 s [6H, (CH3)2N], 3.85 d (1Н, С5Н, J = 10.2 Hz), 4.56 s (1Н, ОН), 6.60 d (2Н, C6H4, J = 8.0 Hz), 6.92 m (2H, C6H4 + 1H, NH), 7.07 s (1H, NH), 10.01 br. s (1Н, NH, OH). Found, %: C 61.83; H 6.81; N 16.91. C17H22N4O3. Calculated, %: C 61.80; H 6.71; N 16.96.

3,6-Dihydroxy-[4-(diethylamino)phenyl]-6-methyl-4,5,6,7-tetrahydro-2H-indazole-5-carboxamide (2f). Yield 35%, mp 258–260°C (ethanol). IR spectrum, ν, cm–1: 3575 (OH), 3404, 3260 (NH), 1660 (CON). 1H NMR spectrum, δ, ppm: 1.08 m [6H, (CH3CH2)2N], 1.24 s (3Н, CH3), 2.30 d (1Н, С4Н, J = 10.2 Hz), 2.56 d (1Н, С7НАНВ, J = 16.0 Hz), 2.60 d (1Н, С7НАНВ, J = 16.0 Hz), 3.30 m [4Н, (CH3CH2)2N], 3.82 d (1Н, С5Н, J = 10.0 Hz), 4.57 s (1Н, ОН), 6.51 d (2H, С6Н4, J = 8.0 Hz), 6.88 d (2Н, С6Н4, J = 8.0 Hz), 6.98 s (1H, NH), 7.13 s (1H, NH), 9.25 br. s (1Н, NH, OH). 13C NMR spectrum, δС, ppm: 12.61, 18.52, 28.88, 35.31, 38.11, 43.52, 56.00, 58.55, 70.21, 100.1, 111.02, 128.81, 129.34, 138.46, 145.76, 158.08, 176.09. Found, %: C 63.63; H 7.41; N 15.72. C19H26N4O3. Calculated, %: C 63.67; H 7.31; N 15.63.

3,6-Dihydroxy-4-(4-fluorophenyl)-6-methyl-4,5,6,7-tetrahydro-2H-indazole-5-carboxamide (2g). Yield 31%, mp 280–282°C (ethanol). IR spectrum, ν, cm–1: 3553 (OH), 3342 (NH), 1628 (CON). 1H NMR spectrum, δ, ppm: 1.26 s (3Н, CH3), 2.34 d (1Н, С4Н, J = 10.0 Hz), 2.55 d (1Н, С7НАНВ, J = 16.5 Hz), 2.62 d (1Н, С7НАНВ, J = 16.4 Hz), 3.96 d (1Н, С5Н, J = 10.0 Hz), 4.58 s (1Н, ОН), 6.95 s (1H, NH), 7.09 m (2H, С6Н4), 7.12 m (2H, С6Н4 + 1H, NH), 10.05 br. s (1Н, NH, OH). Found, %: C 58.92; H 5.34; N 13.82. C15H16FN3O3. Calculated, %: C 59.01; H 5.28; N 13.76.

4-(3-Bromophenyl)-3,6-dihydroxy-6-methyl-4,5,6,7-tetrahydro-2H-indazole-5-carboxamide (2h). Yield 83%, mp 262–264°C (ethanol). IR spectrum, ν, cm–1: 3575 (OH), 3387, 3260 (NH), 1668 (CON). 1H NMR spectrum, δ, ppm: 1.26 s (3Н, CH3), 2.35 d (1Н, С4Н, J = 10.4 Hz), 2.54 d (1Н, С7НАНВ, J = 16.0 Hz), 2.64 d (1Н, С7НАНВ, J = 16.0 Hz), 3.94 d (1Н, С5Н, J = 10.4 Hz), 4.59 s (1Н, ОН), 7.05 s (1H, NH), 7.11 d (1H, С6Н4), 7.22 m (3H, С6Н4 + NH), 7.35 d (1H, С6Н4), 10.25 br. s (1Н, NH, OH). 13C NMR spectrum, δС, ppm: 28.64, 35.39, 38.99, 58.25, 70.09, 99.00, 121.02, 127.52, 128.78, 129.77, 130.70, 138.82, 145.91, 157.87, 175.13. Found, %: C 49.22; H 4.44; N 11.52. C15H16BrN3O3. Calculated, %: C 49.20; H 4.40; N 11.47.

3,6-Dihydroxy-4-(4-chlorophenyl)-6-methyl-4,5,6,7-tetrahydro-2H-indazole-5-carboxamide (2i). Yield 28%, mp 275–277°C (ethanol). IR spectrum, ν, cm–1: 3505 (OH), 3390, 3326 (NH), 1671 (CON). 1H NMR spectrum, δ, ppm: 1.26 s (3Н, CH3), 2.33 d (1Н, С4Н, J = 10.5 Hz), 2.56 d (1Н, С7НАНВ, J = 16.0 Hz), 2.62 d (1Н, С7НАНВ, J = 16.0 Hz), 3.94 d (1Н, С5Н, J = 10.5 Hz), 4.60 s (1Н, ОН), 6.96 s (1H, NH), 7.11 m (2H, С6Н4 + 1H, NH), 7.26 d (2H, С6Н4, J = 8.0 Hz), 10.05 br. s (1Н, NH, OH). Found, %: C 55.92; H 5.14; N 13.12. C15H16ClN3O3. Calculated, %: C 55.99; H 5.01; N 13.06.

Methyl 4-(3,6-dihydroxy-5-carbamoyl-6-methyl-4,5,6,7-tetrahydro-2H-indazol-4-yl)benzoate (2j). Yield 42%, mp 274–276°C (ethanol). IR spectrum, ν, cm–1: 3485 (OH), 3408, 3356 (NH), 1668 (CON). 1H NMR spectrum, δ, ppm: 1.27 s (3Н, CH3), 2.38 d (1Н, С4Н, J = 10.0 Hz), 2.56 d (1Н, С7НАНВ, J = 16.0 Hz), 2.65 d (1Н, С7НАНВ, J = 16.0 Hz), 3.84 s (3H, CH3OCO), 4.03 d (1Н, С5Н, J = 10.0 Hz), 4.64 s (1Н, ОН), 7.00 s (1H, NH), 7.13 s (1H, NH), 7.24 d (2H, С6Н4, J = 8.0 Hz), 7.83 d (2H, С6Н4, J = 8.0 Hz), 9.62 br. s (1Н, NH, OH). 13C NMR spectrum, δС, ppm: 28.64, 35.39, 51.88, 58.05, 70.09, 99.03, 127.40, 128.61, 128.66, 138.73, 148.95, 157.91, 166.33, 175.16. Found, %: C 59.08; H 5.64; N 12.22. C17H19N3O5. Calculated, %: C 59.12; H 5.55; N 12.17.

3,6-Dihydroxy-6-methyl-4-(pyridin-3-yl)-4,5,6,7-tetrahydro-2H-indazole-5-carboxamide (2k). Yield 51%, mp 298–300°C (ethanol). IR spectrum, ν, cm–1: 3550 (OH), 3444, 3340 (NH), 1629 (CON). 1H NMR spectrum, δ, ppm: 1.28 s (3Н, CH3), 2.40 d (1Н, С4Н, J = 10.2 Hz), 2.60 d (1Н, С7НАНВ, J = 16.0 Hz), 2.63 d (1Н, С7НАНВ, J = 16.0 Hz), 3.98 d (1Н, С5Н, J = 10.2 Hz), 4.61 s (1Н, ОН), 6.99 s (1H, NH), 7.16 s (1H, NH); 7.25 d. d (J = 7.8, 4.8 Hz), 7.45 t. d (J = 8.4, 2.0 Hz), 8.32 d (J = 2.2 Hz), 8.37 d. d (J = 4.8, 1.7 Hz) (4H, С5Н4N), 10.98 br. s (1Н, NH, OH). Found, %: C 58.22; H 5.62; N 19.52. C14H16N4O3. Calculated, %: C 58.32; H 5.59; N 19.43.

Antimicrobial activity of compounds 2а, 2b, 2d, 2e, 2g, 2k against Escherichia coli 25922 ATCC and Staphylococcus aureus 6538P ATCC strains was determined by the method of successive dilutions of a solution of the test substances in a meat-peptone broth at a bacterial load of 250 000 microbial units per 1 mL of solution. The maximum dilution leading to the complete suppression of the growth of test microbes was taken as the minimum inhibitory concentration. Dioxidine and furacilin were used as reference drugs.

Analgesic activity was studied by the “hot plate” and “vinegar cramps” methods using outbred mice [37]. In the hot plate test, the test substances were injected intraperitoneally at a dose of 50 mg/kg in 2% starch mucus for 60 min before placing the animals on a metal plate heated to 52°C. An indicator of nociception was the duration of the animal’s stay on the “hot plate” before the onset of a defensive reaction (licking, shaking, jumping) of the hind legs, measured in seconds.

The pain reaction in the “vinegar cramps” test was induced by intraperitoneal injection of 0.75% acetic acid (at the rate of 0.1 mL per 10 g of animal weight) through 30 min after intraperitoneal injection of the test compounds at a dose of 50 mg/kg. During the next 20 min after the injection of acetic acid, the number of writhing was counted for each animal. The control group of animals was injected with an equivalent volume of 2% starch mucus. Metamizole sodium (analgin) at a dose of 50 mg/kg, injected analogously to the tested compounds, was used as a reference. The results were statistically processed using the Student's test. The effect was considered significant at p < 0.05.

The work was performed in compliance with all applicable international, national and institutional guidelines for the care and use of animals.