Design, synthesis, cytotoxicity and 3D-QSAR analysis of new 3,6-disubstituted-1,2,4,5-tetrazine derivatives as potential antitumor agents

2.1 Chemistry

Synthesis of these tetrazine derivatives from both series was achieved according to Scheme 1, using tetrazine 6 (Coburn et al., 1991), as the starting material, and simple well-known organic reactions at room temperature. To obtain compounds from series I (1a-l), phenyl-piperazine derivatives 5a-l were prepared from corresponding aryl chloride 3a-l. Nucleophilic substitution of 3a-l by tert-butyl-4-aminopiperidine-1-carboxylate, in a mixture of THF and CH₂Cl₂ (1:1), furnished Boc-derivatives 4a-l (65–83%). Deprotection reactions from these Boc-derivatives were carried out with trifluoroacetic acid (TFA), yielding the free-amines 5a-l. Without further purification, these free-amines were reacted with 6 in acetonitrile as a solvent, and in the presence of triethylamine (TEA), yielded target compounds 1a-l (40–69%) (Rusinov et al., 2006). Such low yields in the last step could be related to the work-up and cleansing stages (including chromatographic separations). Similarly, compounds belonging to series II (2a-j) were synthesized through the nucleophilic substitution of 6 by commercially available aryl-piperazines in acetonitrile as solvent with TEA present. Yields from this step were higher than the other analogues (52–84%), probably due to the purity and stability of the nucleophiles. Target compound chemical structures from both series were established by their spectral properties (IR, ¹H NMR, ¹³C NMR and HRMS, see Section 4 and Electronic supplementary information, ESI) and for compounds 1g, 2a, 2c and 2h, by X-ray crystal structure analysis.

Close

Scheme 1 Reagents and conditions: (i) tert-Butyl-4-aminopiperidine-1-carboxylate, THF/CH₂Cl₂, rt, 12 h, 65–83%; (ii) TFA, rt, 30 min; (iii) 6, TEA (excess), acetonitrile, rt, overnight, 30–69%; (iv) Arylpiperazines or arylpiperidines, TEA (excess), acetonitrile, rt, overnight, 52–84%.

Export to PPT

2.2 Crystallographic studies

Molecular structures from the four selected tetrazine derivatives 1g, 2a, 2c and 2h were determined by X-ray crystallography (Fig. 2). Selected distances, angles and dihedral angles are summarized in Table S-1 (see Electronic supplementary information). All four tetrazines had similar molecular structure. Bond distances and angles were in the typical range of similar organic systems (Patra et al., 2004). Tetrazines exhibit a bending point in the piperazine ring with dihedral angles of −111.1(3)° (C7-N7-C8-C9, for tetrazine 1g), −116.3(2)° (C7-N1-C14-C13, for tetrazine 2a), −125.6(0)° (C5-N7-C6-C7, for tetrazine 2c) and −103.8(3)° (C7-N7-C10-C11, for tetrazine 2h), due to the chair shape adopted by the piperazine portion of the molecule. The pyrazole group is rotated slightly out of the plane of the tetrazine ring, showing dihedral angles of 24.0(4)° (N1-N2-C6-N3, for tetrazine 1g), 32.5(2)° (N6-N5-C8-N8, for tetrazine 2a), 28.6(7)° (N2-N1 C4-N6, to the tetrazine 2c) and 33.2(3)° (N1-N2-C6-N3, for tetrazine 2h). Tetrazine 1g exhibits a rotation out of the plane of the carbonyl group with respect to phenyl ring (−49.5(4)° (C15-C14-C13-O1)) and in the tetrazine 2h a coplanarity is shown between the piperazine and the pyridine group (11.4(3)° C11-N8-C12-N9)).

Close

Fig. 2 Molecular structure of selected tetrazines 1g, 2a, 2c and 2h. Thermal ellipsoids are shown with 30% probability.

Export to PPT

2.3 Cytotoxic studies

We studied the in vitro cytotoxic effect of the previously mentioned compounds on H1975, HL-60, HCT116 and HeLa cancer cell lines and no-neoplastic Vero cells. Conventional colorimetric assay was used to estimate the IC₅₀ values, representing the concentration of a drug that is needed for 50% inhibition in vitro after 72 h of continuous exposure to compounds. Triplicate tests using four serial dilutions (from 0.05 to 50 µM) were done for each sample, using the drugs etoposide and vatalanib as a positive control.

Table 1 shows the IC₅₀ cytotoxicity values for compounds 1a-l, 2a-j and 6. In general, tetrazine derivatives showed to be heterogeneous, but in the most cases the unsymmetrical tetrazines were more potent than the symmetrical starting material (6, with IC₅₀ values on all cancer cell lines >50 µM). In a general analysis of cytotoxicity, HCT116 cells seemed to be more resistant (all compounds tested with IC₅₀ value >5 µM). H1975, HL60, Hela and Vero cells had variable sensitivity. However, after further cytotoxicity analysis for each cancer cell line, we conclude that:

1. For HCT116 cells, all compounds were less active than etoposide. On the contrary, compounds 1b, 1c, 1h, 1i, 1j and 1l showed higher activity than vatalanib. All derivatives were less selective than etoposide and more selective than vatalanib (SI value = 0.3).

2. For HeLa cells, compounds 1f and 1h (IC₅₀ values = 6.6 and 4.1 µM, respectively) were more potent than etoposide, and exhibited better or equal selectivity than the same reference compound (SI values = 7.6 and 3.2). In addition, compounds 1b, 1c and 1j showed similar activity, but a higher selectivity compared to etoposide (SI values = 5.9, 5.4, and 7.6, except 1j with a SI value < 3.0). It is important to mention that all active compounds (IC₅₀ values < 11 µM) were more potent and selective than vatalanib.

3. For HL60 cells, these were our best results in potency and selectivity from some of the tetrazine derivatives compared to etoposide and vatalanib. Specifically, compounds 1h-l elicited the lowest IC₅₀ values in this study (<1.9 µM). Interestingly, 1i and 1j were five to six times more selective than etoposide and more than one hundred times more selective than vatalanib (SI values = 27 and 19). 1i and 1j are the most promising compounds for further biological studies.

4. For H1975 cells, compounds 1k, 1l and 2h exhibited from higher to similar potency than etoposide (IC₅₀ values = 2.4, 4.4 and 7.4 µM, respectively), but only 2h showed a higher selectivity (SI value = 6.8). Moreover, compounds 1a, 1i, 1j, 1k, 1l and 2h were more potent and selective than vatalanib (IC₅₀ values < 20 µM and SI values > 0.5).

On the other hand, antitumor drugs should exhibit low toxicity in mammalian host cells, and thus, more selective compounds are very promising for the development of new antitumor agents. These results are in agreement with the National Cancer Institute (NCI) protocols, which consider active compounds exhibiting IC₅₀ values < 10 μM or 15 μM (NCI/NIH, 2014).

2.4 Structure activity relationship (SAR)

The SAR analysis depends on the comparison between the cytotoxic activity against cancer cell lines and structural variations from compounds of the series I or II. Thus, a look at the IC₅₀ values for each cancer cell line suggests that from a chemical point of view, there are interesting structural features worth considering.

Firstly, in general, compounds from series I were more potent than series II. This evidence could indicate, that amide moiety in the compounds of series I, is an important linker between the aromatic ring and the heterocyclic system, and is the major structural difference with the compounds from the series II.

Secondly, some substitutions on the phenyl moiety of 1a, led us to understand the scope of some chemical modifications. This was especially notable in some cases where there existed a clear behaviour on the four cancer cell lines. For example, when the benzene ring was substituted in ortho-position with an electron-donating group (methyl for 1g, IC₅₀ values > 50 µM), this modification did not generate an increase in the cytotoxic activity on four cancer cell lines. However, if this substituent was a bromine atom (1h), the cytotoxicity increased significantly (IC₅₀ values < 5 µM), except on H1975 cells. On the other hand, a 2,4-disubstitution (1d, IC₅₀ values > 50 µM) was unsuccessful at obtaining a compound with better activity compared with 1a. Regarding compounds with substitution on meta-positions, 1j exhibited a significant improvement in potency against all cell panels (IC₅₀ values = 0.7–13 µM). Nevertheless, in the compounds that have a 3,4- or 3,5-disubstitution pattern (1e or 1k and 1b or 1c, respectively), the effect on the cytotoxicity depends on the combination of the stereoelectronic features of these groups. A similar behaviour was observed for the para-substitution, and this is because when an electron-withdrawing group was incorporated (cyano group in 1f), the cytotoxic activity did not change compared to 1a, except in HeLa cells. However, when substitution was done by a chloromethyl group (1l), diminished IC₅₀ values were observed in all cell panels (<11 µM).

In a third observation, when comparing 1a with 1i, the IC₅₀ values indicated that replacement of the benzene ring by a naphthalene ring was relevant for an increase in cytotoxicity against HCT116 cells and very significant on Hela cells. On H1975 cells, this modification slightly enhanced cytotoxicity, and on HL-60 cells, it was unsuccessful.

For the less active tetrazine derivatives in this study (series II), the influence of the substituents in the phenyl group, on the increase in cytotoxicity, was not achieved when reference compound 2a was modified. Only two compounds exhibited a significant potency and selectivity; 2g on HL-60 cells and 2h on H1975 cells (IC₅₀ values of 8.1 and 7.4 µM, and SI values of 6.2 and 6.8, respectively). These results indicate that, on HL-60 cell, the addition of an extra nitrogen atom and their position in the aromatic ring, increased the cytotoxic activity (2f-h, IC₅₀ values < 29 µM). But, if in this heterocyclic moiety, an electron-withdrawing group or an extra nitrogen atom is incorporated, the cytotoxicity is reduced (2h-j, IC₅₀ values > 29 µM).

Finally, the SAR analysis mentioned above, provides information about the effectivity of chemical modification for these tetrazine derivatives on the cytotoxic activity in cancer cell lines. Furthermore, 3D-QSAR gives an even more precise explanation of the cytotoxicity differences of the studied compounds. These separate 3D-QSAR results and discussions are given in the following section.

2.5 3D-QSAR

From the works of Cramer and Klebe (Cramer et al., 1988; Klebe et al., 1994), comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) are useful methodologies to understand the pharmacological properties of a series of compounds. The steric and electrostatic maps obtained help to (a) understand the nature of ligand-receptor interactions; (b) predict biological affinity and (c) aid in the rational design of new promising compounds. 3D-QSAR studies were performed on the four cancer cell lines with compounds that had IC₅₀ values < 50 µM. Information related to steric (S), electrostatic (E) and hydrophobic (H) properties was concordant among the models. In the CoMSIA studies, the donor and acceptor fields were not related to the observed activity. Models exhibited good r² > 0.8, small standard error of estimation analysis (SEE) of non-cross-validation, as well as high F-test values (F). 3D-QSAR analysis, from the results of the cytotoxicity in HL60 cells gave the most extensive information about the chemical requirements of tetrazine derivatives. In Table 2, we report the step-by-step search to find the best CoMFA and CoMSIA field combinations in the HL60 model. The criterion to select the best models was the LOO cross validated r² (q²). Compounds exhibited a good linear correlation between their actual versus predicted pIC₅₀, for both CoMFA and CoMSIA models (Fig. 3), and the residual value was less than 0.25. The experimental versus calculated biological activity is shown in Table 3.

Close

Fig. 3 CoMFA and CoMSIA experimental versus predicted pIC₅₀ applied to tetrazine derivative cytotoxicity on HL60 cells.

Export to PPT

CoMFA and CoMSIA contour maps for HL60, HCT116 and H1975 shows a large green polyhedron around the aromatic ring at the end of the most active compounds, and a yellow polyhedron around the piperazine and piperidine in the less active compounds (Fig. 4(A, B), 5(A, B), S1 (A, B) and S3 (A)). This suggests that bulky groups such as sulphonamide in compound 1k, are highly favourable for antitumor activity. In fact, the most active compounds usually carry halogens or a bulky naphthalene ring at the end. According to the HL60 Models, the best positions for inserting bulky groups should be positions 3, 4 and 5 of the benzene ring. However, the presence of groups such as halogens or methyl in the compounds 1b-c, e-f, implicates that a higher steric hindrance is necessary in these positions. Use of other non-planar bulky rings such as adamantyl ring, would be interesting to explore too. The less active compounds do not have the amide linker between piperidine or piperazine and the phenyl ring, and these molecules point their terminal rings to the yellow polyhedron. So, lengthening of the chains is beneficial for activity. CoMSIA Hydrophobic contour maps (Figs. 5(E, F), S1 and S2(E,F)) complement that, the hydrophobic favourable yellow polyhedra are around the halogens and phenyls in the most active derivatives. Only the contour maps in H1975 (Fig. S3(B)) show a predominance of hydrophilic polyhedra around the terminal ring of the compounds. A white polyhedron is depicted around the nitrogen atom of the amide group. Thus, a reasonable modification would be the replacement of the amide by an amine protonable nitrogen.

Close

Fig. 4 3D-QSAR in HL60. CoMFA Steric (A, B) and electrostatic (C, D) contour maps around the most active compounds 1i, 1j, 1k (left) and the less active compounds 2d, 2i and 2j (right). The green contours mean that the presence of bulky groups favours biological activity, and in yellow, small substituents are also favourable. In red, electron-withdrawing substituents favour activity, and in blue electropositive substituents are favourable.

Export to PPT

Close

Fig. 5 CoMSIA in HL60. A and B are steric contour maps. C and D are electrostatic contour maps. E and F are hydrophobic contour maps of the most active compounds (1i, 1j and 1k (on the left)) and the least active (2d, 2i and 2j (on the right)). Green means that the presence of bulky groups favours biological activity, whereas yellow indicates that the use of small groups is also favourable. Red means that electronegative groups favour biological activity, and blue means that electropositive groups are also favourable. On hydrophobic maps (E and F), yellow means that using lipophilic substituents favours biological activity, while white polyhedrons mean that using hydrophilic substitutes is also favourable.

Export to PPT

On the other hand, the CoMFA and CoMSIA electrostatic contour maps show blue polyhedra around the peripheral phenyl moiety. That means the benzene ring should be substituted with electron withdrawing groups. Therefore, the use of a ketone instead of an amide linker would be beneficial. The replacement of the benzene ring for pyridine or their derivatives in series I should be explored. Only in the CoMFA electrostatic in HL60, two prominent red polyhedra are depicted near the carbonyl group in the amide of the most active compounds and surrounding the pyridine in the less active compounds. This suggests that a way to bolster the activity of these less active molecules is to replace the pyridine ring for activated phenyl rings.

2.6 Cell death mechanism assay

HCT116, HeLa, HL60 and H1975 cells lines were incubated for 16 h in the presence of 50 µM of the most promising tetrazine derivatives (compounds with the lowest IC₅₀ values). By flow cytometry analysis, it was determined a population of viable cells impermeable to PI (propidium iodide; PI negative) and two populations of PI-permeable (PI positive) dead cells which were distinguished based on fluorescence intensity to either hypodiploid apoptotic cells or necrotic cells with intact DNA. As positive control, the same quantity of etoposide and vatalanib was used. The results are shown in Fig. 6. HCT116 cells shown both apoptosis and necrosis, being apoptosis the main cell death mechanism. A 40% of apoptosis was produced by 1c and 1i, with a similar behaviour than etoposide. In HL60 cells, cell death by necrosis is the main cell death mechanism. In Hela cells, it was observed a slow increase in apoptotic and necrotic cell death for all compounds studied in this cell line. H1975 cells shown both cell death mechanisms with an increase in necrosis in two compounds (1j, 1l). In Vero cells, the main cell death is necrosis with the compound 1l.

Close

Fig. 6 Viability in different cancer cells lines treated with selected tetrazines. Cells were stimulated for 16 h with the different compounds (50 µM). The cells were then stained with propidium iodide (PI; 10 µg/ml) and cell viability and cell death by apoptosis or necrosis was evaluated by flow cytometry.

Export to PPT

4.1 Materials and measurements

Melting points were determined on a Kofler Thermogerate apparatus and were uncorrected. Infrared spectra were recorded on a JASCO FT/IR-400 spectrophotometer. Nuclear magnetic resonance (NMR) spectra were recorded, unless otherwise specified, on a Bruker AM-400 instrument using deuterated chloroform or dimethylsulphoxide solutions containing tetramethylsilane as an internal standard. Mass spectra were obtained on a HP 5988A mass spectrometer. HRMS-ESI-MS experiments were performed using a Thermo Scientific Exactive Plus Orbitrap spectrometer with a constant nebulizer temperature of 250 °C. The experiments were carried out in positive or negative ion mode, with a scan range of m/z 300.00–1510.40 with a resolution of 140,000. The samples were infused directly into the ESI source, via a syringe pump, at flow rates of 5 µL min⁻¹, through the instrument’s injection valve. Thin layer chromatography (tlc) was performed using Merck GF-254 type 60 silica gel. Column chromatography was carried out using Merck type 9385 silica gel. The purity of the compounds was determined by tlc, NMR spectra and HRMS. For X-ray crystal structure analysis, data sets were collected with a STOE IPDS II two-circle-diffractometer using Mo Kα radiation (λ = 0.71073 Å). The intensities were corrected for absorption by an empirical correction with X-Area. (Stoe&Cie, 2001). The structures were solved by direct methods (SHELXS) (Sheldrick, 2008) and refined by full-matrix least-squares calculations on F² (SHELXL-97). Anisotropic displacement parameters were refined for all non-hydrogen atoms. Selected crystallographic data are listed in Table S-1 (ESI).

4.2 Synthesis and characterization

4.2.1

4.2.1 General synthetic procedure of compounds 4a-l

tert-Butyl-4-aminopiperidine-1-carboxylate (N-Boc-4-aminopiperidine) (1 mmol) and the corresponding aryl chloride 3a-l (1 mmol), were added to a reaction flask that contained dry THF and CH₂Cl₂ (1:1) and a small portion of molecular sieves. The mixture was stirred at room temperature overnight. Then the mixture was put in ice/water and diethyl ether (50 mL) was added. The organic extracts were washed with two portions of Na₂CO₃ (2 × 20 mL, 10%) and water (2 × 20 mL), and dried over Na₂SO₄. The solution was evaporated to dryness and the crude product was purified by column chromatographic on silica gel using dichloromethane as eluent.

Close

Export to PPT

4.2.1.1

4.2.1.1 tert-Butyl-4-benzamidopiperidine-1-carboxylate (4a)

Yield, 70%. White solid, mp 160–161 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.56 (d, J = 7.7 Hz, 2H, H2 + H6), 7.24 (m, 3H, H3, H4 + H5), 5.94 (d, J = 7.4 Hz, 1H, NH), 3.98–3.90 (m, 3H, N-CH-pip + N-CH₂-pip), 2.71 (t, J = 12.2 Hz, 2H, N-CH₂-pip), 1.82 (d, J = 11.7 Hz, 2H, -CH₂-pip), 1.27 (m, 11H, -CH₂-pip + 3 × CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 166.91 (C⚌O), 154.75 (O—C⚌O), 134.55 (C1), 131.52 (C4), 128.58 (C3 + C5), 126.89 (C2 + C6), 79.73 (O—C), 47.25 (C4″), 42.79 (C2″ + C6″), 32.14 (C3″ + C5″), 28.44 (3 × CH₃). IR (KBr, cm⁻¹): 3278, 2999, 1686, 1628, 1276, 696. HRMS for (C₁₇H₂₄N₂O₃ [M⁻]). Calcd: 303.1714. Found: 303.1711.

4.2.1.2

4.2.1.2 tert-Butyl-4-[(3,5-dimethylbenzoyl)amino]piperidine-1-carboxylate (4b)

Yield, 67%. White solid, mp 108–109 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.23 (s, 2H, H2 + H6), 6.99 (s, 1H, H4), 6.04 (d, J = 7.5 Hz, 1H, NH), 3.96 (d, J = 11.6 Hz, 3H, N-CH-pip + N-CH₂-pip), 2.77 (t, J = 12.4 Hz, 2H, N-CH₂-pip), 2.22 (s, 6H, 2 × CH₃), 1.87 (d, J = 12.2 Hz, 2H, -CH₂-pip), 1.38–1.23 (m, 11H, -CH₂-pip + 3 × CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 167.24 (C⚌O), 154.72 (O—C⚌O), 138.23 (C3 + C5), 134.58 (C1), 133.03 (C4), 124.66 (C2 + C6), 79.64 (O—C), 47.12 (C4″), 42.77 (C2″ + C6″), 32.12 (C3″ + C5″), 28.43 (3 × CH₃), 21.20 (2 × CH₃). IR (KBr, cm⁻¹): 3253, 2930, 1689, 1633, 1313, 936, 726. HRMS for (C₁₉H₂₈N₂O₃ [M⁻]). Calcd: 331.2027. Found: 331.2026.

4.2.1.3

4.2.1.3 tert-Butyl-4-[(3-chloro-5-fluorobenzoyl)amino]piperidine-1-carboxylate (4c)

Yield, 77%. White solid, mp 129–131 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.38 (s, 1H, H2), 7.24 (d, J = 8.7 Hz, 1H, H4), 7.05 (d, J = 8.0 Hz, 1H, H6), 6.21 (d, J = 7.4 Hz, 1H, NH), 3.94 (d, J = 5.4 Hz, 3H, N-CH-pip + N-CH₂-pip), 2.72 (t, J = 12.2 Hz, 2H, N-CH₂-pip), 1.83 (d, J = 11.9 Hz, 2H, -CH₂-pip), 1.27 (m, 11H, -CH₂-pip + 3 × CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 164.34 (d, J_CF = 2.4 Hz, C⚌O), 162.54 (d, J_CF = 251.6 Hz, C5), 154.70 (O—C⚌O), 137.76 (d, J_CF = 7.5 Hz, C3), 135.49 (d, J_CF = 10.0 Hz, C1), 123.12 (d, J_CF = 3.2 Hz, C2), 119.07 (d, J_CF = 24.8 Hz, C4), 112.98 (d, J_CF = 22.9 Hz, C6), 79.84 (O—C), 47.64 (C4″), 42.77 (C2″ + C6″), 31.97 (C3″ + C5″), 28.42 (3 × CH₃). ¹⁹F NMR (376 MHz, CDCl₃) δ -109.55. IR (KBr, cm⁻¹): 3253, 3091, 1691, 1588, 1283, 1140, 738. HRMS for (C₁₇H₂₂ClFN₂O₃ [M⁻]). Calcd: 355.1230. Found: 355.1226.

4.2.1.4

4.2.1.4 tert-Butyl-4-{[2-nitro-4-(trifluoromethyl)benzoyl]amino} piperidine-1-carboxylate (4d)

Yield, 80%. White solid, mp 150–152 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.43 (s, 1H, H3), 8.17 (d, J = 4.7 Hz, 1H, H5), 7.97 (d, J = 8.0 Hz, 1H, H6), 6.63 (s, 1H, NH), 4.32 (m, 1H, N-CH-pip), 3.54 (s, 2H, N-CH₂-pip), 2.47 (t, J = 8.1 Hz, 2H, N-CH₂-pip), 2.23 (dd, J = 15.3, 7.6 Hz, 2H, -CH₂-pip), 1.75–1.67 (m, 2H, -CH₂-pip), 1.55 (s, 9H, 3 × CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 165.63 (C⚌O), 154.74 (O—C⚌O), 146.15 (C2), 135.33 (C1), 133.58 (d, J _CF = 34.5 Hz, C4), 130.72 (C6), 129.75 (q, J_CF = 3.3 Hz, C3), 126.41 (CF₃), 121.10 (d, J _CF = 3.8 Hz, C5), 77.22 (O—C), 47.37 (C4″), 44.62 (C2″ + C6″), 43.61 (C3″ + C5″), 27.68 (3 × CH₃). ¹⁹F NMR (376 MHz, CDCl₃) δ −63.10. IR (KBr, cm⁻¹): 3293, 1695, 1643, 1551, 1421, 1180. HRMS for (C₁₈H₂₂F₃N₃O₅ [M⁻]). Calcd: 416.1433. Found: 416.1440.

4.2.1.5

4.2.1.5 tert-Butyl-4-[(3-fluoro-4-methoxybenzoyl)amino]piperidine-1-carboxylate (4e)

Yield, 74%. White solid, mp 177–179 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.49 (br.s, 2H, H2 + H6), 6.90 (t, J = 8.2 Hz, 1H, H5), 6.19 (d, J = 7.5 Hz, 1H, NH), 4.04 (d, J = 11.6 Hz, 3H, N-CH-pip + N-CH₂-pip), 3.87 (s, 3H, OCH₃), 2.83 (t, J = 12.6 Hz, 2H, N-CH₂-pip), 1.94 (d, J = 12.2 Hz, 2H, -CH₂-pip), 1.46–1.29 (m, 11H, -CH₂-pip + 3 × CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 165.31 (C⚌O), 154.71 (O—C⚌O), 151.86 (d, J_CF = 247.4 Hz, C3), 150.38 (d, J_CF = 10.7 Hz, C4), 127.29 (d, J_CF = 5.4 Hz, C1), 123.49 (d, J_CF = 3.5 Hz, C6), 115.06 (d, J_CF = 19.7 Hz, C2), 112.62 (d, J_CF = 1.8 Hz, C5), 79.71 (O—C), 56.26 (OCH₃), 47.34 (C4″), 42.79 (C2″ + C6″), 32.10 (C3″ + C5″), 28.42 (3 × CH₃). ¹⁹F NMR (376 MHz, DMSO-d₆) δ −134.96. IR (KBr, cm⁻¹): 3384, 2947, 1692, 1632, 1281, 1019, 764. HRMS for (C₁₈H₂₅FN₂O₄ [M⁻]). Calcd: 351.1726. Found: 351.1723.

4.2.1.6

4.2.1.6 tert-Butyl-4-[(4-cyanobenzoyl)amino]piperidine-1-carboxylate (4f)

Yield, 71%. White solid, mp 182–183 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.99 (d, J = 7.9 Hz, 2H, C3″ + C5″), 7.71 (d, J = 7.8 Hz, 2H, C2″ + C6″), 6.40 (d, J = 7.0 Hz, 1H, NH), 4.10 (m, 3H, N-CH-pip + N-CH₂-pip), 2.87 (s, 2H, N-CH₂-pip), 1.94 (d, J = 12.0 Hz, 2H, -CH₂-pip), 1.45 (s, 11H, -CH₂-pip + 3 × CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 165.07 (C⚌O), 154.60 (O—C⚌O), 138.68 (C1), 131.86 (C3 + C5), 128.17 (C2 + C6), 118.09 (CN), 115.01 (C4), 79.35 (O—C), 47.41 (C4″), 42.73 (C2″ + C6″), 31.41 (C3″ + C5″), 28.27 (3 × CH₃). IR (KBr, cm⁻¹): 3359, 2982, 2230, 1693, 1641, 1430, 1302, 864, 769. HRMS for (C₁₈H₂₃N₃O₃ [M + H]+). Calcd: 328.1667. Found: 328.1663.

4.2.1.7

4.2.1.7 tert-Butyl-4-[(2-methylbenzoyl)amino]piperidine-1-carboxylate (4g)

Yield, 83%. White solid, mp 103–104 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.33 (d, J = 6.8 Hz, 2H, H4 + H6), 7.22 (d, J = 7.5 Hz, 2H, H3 + H5), 7.08 (s, 1H, NH), 4.10 (m, 3H, N-CH-pip + N-CH₂-pip), 2.92 (s, 2H, N-CH₂-pip), 2.44 (s, 3H, CH₃), 1.99 (d, J = 12.3 Hz, 2H, -CH₂-pip), 1.48 (s, 11H, -CH₂-pip + 3 × CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 169.52 (C⚌O), 154.56 (O—C⚌O), 136.96 (C1), 135.49 (C2), 130.56 (C4), 129.39 (C3), 126.80 (C6), 125.46 (C5), 79.35 (O—C), 46.80 (C4″), 42.31 (C2″ + C6″), 31.65 (C3″ + C5″), 28.30 (3 × CH₃), 19.48 (CH₃). IR (KBr, cm⁻¹): 3271, 2929, 1692, 1634, 1429, 1145, 740. HRMS for (C₁₈H₂₆N₂O₃ [M⁻]). Calcd: 317.1871. Found: 317.1870.

4.2.1.8

4.2.1.8 tert-Butyl-4-[(2-bromobenzoyl)amino]piperidine-1-carboxylate (4h)

Yield, 75%. White solid, mp 64–65 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.53 (d, J = 7.9 Hz, 1H, H6), 7.47 (d, J = 7.0 Hz, 1H, H3), 7.31 (t, J = 7.0 Hz, 1H, H4), 7.23 (t, J = 7.5 Hz, 1H, H5), 5.95 (s, 1H, NH), 4.06 (m, 3H, N-CH-pip + N-CH₂-pip), 2.90 (t, J = 11.5 Hz, 2H, N-CH₂-pip), 2.00 (d, J = 11.6 Hz, 2H, -CH₂-pip), 1.42 (s, 11H, -CH₂-pip + 3 × CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 166.94 (C⚌O), 154.69 (O—C⚌O), 137.74 (C1), 133.33 (C3), 131.29 (C5), 129.59 (C4), 127.62 (C6), 119.20 (C2), 79.72 (O—C), 47.42 (C4″), 42.31 (C2″ + C6″), 31.81 (C3″ + C5″), 28.43 (3 × CH₃). IR (KBr, cm⁻¹): 3242, 3066, 1689, 1591, 1431, 743. HRMS for (C₁₇H₂₃BrN₂O₃ [M + H]⁺). Calcd: 381.0819. Found: 381.0825.

4.2.1.9

4.2.1.9 tert-Butyl-4-(2-naphthoylamino)piperidine-1-carboxylate (4i)

Yield, 82%. White solid, mp 156–158 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.22 (s, 1H, H2), 7.92–7.78 (m, 4H, H3, H6, H7, H8), 7.62–7.41 (m, 2H, H4 + H5), 6.27 (s, 1H, NH), 4.22–4.08 (m, 3H, N-CH-pip + N-CH₂-pip), 2.87 (t, J = 12.1 Hz, 2H, N-CH₂-pip), 2.01 (d, J = 11.0 Hz, 2H, -CH₂-pip), 1.55–1.35 (m, 11H, -CH₂-pip + 3 × CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 166.94 (C⚌O), 154.75 (O—C⚌O), 134.73 (C1), 132.60 (C2a), 131.79 (C6a), 128.88 (C3), 128.46 (C5), 127.75 (C2), 127.67 (C7), 127.33 (C6), 126.79 (C4), 123.57 (C8), 79.72 (O—C), 47.37 (C4″), 42.78 (C2″ + C6″), 32.18 (C3″ + C5″), 28.45 (3 × CH₃). IR (KBr, cm⁻¹): 3267, 2951, 1701, 1685, 1307, 778. HRMS for (C₂₁H₂₆N₂O₃ [M⁻]). Calcd: 353.1865. Found: 353.1871.

4.2.1.10

4.2.1.10 tert-Butyl-4-[(3-chlorobenzoyl)amino]piperidine-1-carboxylate (4j)

Yield, 65%. White solid, mp 135–137 °C. 1H NMR (400 MHz, CDCl3) δ 7.62 (s, 1H, H2), 7.51 (d, J = 7.7 Hz, 1H H4), 7.34 (d, J = 8.1 Hz, 1H, H6), 7.23 (t, J = 7.8 Hz, 1H, H5), 6.12 (d, J = 7.6 Hz, 1H, NH), 4.09–3.87 (m, 3H, N-CH-pip + N-CH₂-pip), 2.76 (t, J = 12.4 Hz, 2H, N-CH₂-pip), 1.96–1.82 (m, 2H, -CH₂-pip), 1.41–1.21 (m, 11H, -CH₂-pip + 3 × CH₃). ¹³C NMR (101 MHz, CDCl3) δ 165.54 (C⚌O), 154.71 (O—C⚌O), 136.35 (C1), 134.70 (C3), 131.50 (C4), 129.88 (C5), 127.28 (C6), 125.08 (C2), 79.76 (O—C), 47.46 (C4″), 42.78 (C2″ + C6″), 32.04 (C3″ + C5″), 28.43 (3 × CH₃). IR (KBr, cm⁻¹): 3273, 2979, 1795, 1692, 1428, 1304, 750. HRMS for (C₁₇H₂₃ClN₂O₃ [M⁻]). Calcd: 337.1324. Found: 337.1326.

4.2.1.11

4.2.1.11 tert-Butyl-4-(4-chloro-3-sulfamoylbenzamido)piperidine-1-carboxylate (4k)

Yield, 77%. Brown oil. ¹H NMR (400 MHz, DMSO-d₆) δ 7.75 (d, J = 3.6 Hz, 1H, H6), 7.53 (d, J = 4.9 Hz, 1H, H4), 7.07 (t, J = 4.3 Hz, 1H, NH), 6.13 (s, 1H, H3), 4.84 (br.s, 2H, NH₂), 4.36–4.17 (m, 1H, N-CH-pip), 3.37 (t, J = 11.8 Hz, 2H, N-CH₂-pip), 2.13 (d, J = 10.2 Hz, 2H, N-CH₂-pip), 1.74 (d, J = 8.5 Hz, 2H, -CH₂-pip), 1.25 (br.s, 11H, -CH₂-pip + 3 × CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 163.64 (C⚌O), 161.55 (O—C⚌O), 134.78 (C3), 132.01 (C4), 130.00 (C1), 128.45 (C2), 127.60 (C5), 127.42 (C6), 79.19 (O—C), 47.02 (C4″), 44.36 (C2″ + C6″), 31.53 (C3″ + C5″), 28.22 (3 × CH₃). IR (KBr, cm⁻¹): 3349, 2952, 1672, 1637, 1531, 1431, 736. HRMS for (C₁₇H₂₄ClN₃O₅S [M⁻]). Calcd: 416.1052. Found: 416.1112.

4.2.1.12

4.2.1.12 tert-Butyl-4-{[4-(chloromethyl)benzoyl]amino}piperidine-1-carboxylate (4l)

Yield, 65%, white solid; mp: 159–162 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.96 (d, J = 7.9 Hz, 2H, H2 + H6), 7.38 (d, J = 7.9 Hz, 2H, H3 + H5), 4.55 (br, 2H, CH₂), 4.02 (m, 3H, N-CH-pip + N-CH₂-pip), 2.81 (t, J = 12.8 Hz, 2H, N-CH₂-pip), 1.97 (d, J = 11.1 Hz, 2H, -CH₂-pip), 1.42 (s, 11H, -CH₂-pip + 3 × CH₃). ¹³C NMR (50 MHz, DMSO-d₆) δ 162.65 (C⚌O), 161.15 (O—C⚌O), 136.59 (C4), 129.49 (C1), 125.61 (C3 + C5), 123.06 (C2 + C6), 74.20 (O—C), 41.98 (C—Cl), 40.14 (C4″), 37.47 (C2″ + C6″), 26.51 (C3″ + C5″), 23.09 (3 × CH₃). IR (KBr, cm⁻¹): 3312, 2971, 1690, 1636, 1425, 711. HRMS for (C₁₈H₂₅ClN₂O₃ [M⁻]). Calcd: 351.1481. Found: 351.1482.

4.2.2

4.2.2 General synthetic procedure of compounds 1a-l

The protective Boc groups were removed from the 4a-l compounds using TFA (Trifluoroacetic acid) (15 mmol) and TES (Triethylsilane) (3 mmol) as solvent at room temperature for 30 min. Once TFA and TES are removed, the free amine 5a-l obtained was used without further purification. Finally, this amine (1 mmol) was mixed with tetrazine 6 (1 mmol) in acetonitrile (40 mL), an excess of TEA (3 mmol) and was stirred overnight at room temperature. After completion of the reaction, the mixture was concentrated. The solid residue was purified by column chromatography on silica gel using CH₂Cl₂/Acetone (5:1) as eluent to yield 1a-l.

Close

Export to PPT

4.2.2.1

4.2.2.1 N-(1-(6-(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetrazin-3-yl)piperidin-4-yl)benzamide (1a)

Yield, 30%. Red solid, mp 172–174 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.60 (d, J = 5.0 Hz, 2H, H2 + H6), 7.31–7.23 (m, 3H, H3, H4 + H5), 6.25 (d, J = 6.2 Hz, 1H, NH), 5.91 (s, 1H, H4′), 4.71 (d, J = 11.2 Hz, 2H, N-CH₂-pip), 4.19 (s, 1H, N-CH-pip), 3.13 (d, J = 9.9 Hz, 2H, N-CH₂-pip), 2.38 (d, J = 2.2 Hz, 3H, CH₃), 2.14 (d, J = 2.5 Hz, 3H, CH₃), 2.06 (d, J = 9.2 Hz, 2H, -CH₂-pip) 1.44 (d, J = 11.0 Hz, 2H, -CH₂-pip). ¹³C NMR (101 MHz, CDCl₃) δ 167.04 (C⚌O), 160.25 (C4″), 156.68 (C1″), 152.04 (C5′), 141.89 (C3′), 134.35 (C1), 131.59 (C4), 128.56 (C3 + C5), 126.96 (C2 + C6), 109.61 (C4′), 47.11 (C4 ${}^{‴}$ ), 42.94 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 31.64 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 13.68 (CH₃), 13.44 (CH₃). IR (KBr, cm⁻¹): 3274, 1629, 1556, 1534. HRMS for (C₁₉H₂₃N₈O [M + H]⁺). Calcd: 379.1995. Found: 379.1989.

4.2.2.2

4.2.2.2 N-(1-(6-(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetrazin-3-yl)piperidin-4-yl)-3,5-dimethylbenzamide (1b)

Yield, 34%. Red solid, mp 205–207 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.23 (s, 2H, H2 + H6), 6.97 (s, 1H, H4), 6.22 (d, J = 7.6 Hz, 1H, NH), 5.95 (s, 1H, H4′), 4.75 (d, J = 13.7 Hz, 2H, N-CH₂-pip), 4.30–4.14 (m, 1H, N-CH-pip), 3.27–3.12 (m, 2H, N-CH₂-pip), 2.42 (s, 3H, CH₃), 2.19 (s, 9H, 3 × CH₃), 2.13–2.07 (m, 2H, -CH₂-pip), 1.53–1.43 (m, 2H, -CH₂-pip). ¹³C NMR (101 MHz, CDCl₃) δ 167.45 (C⚌O), 160.25 (C4″), 156.66 (C1″), 152.01 (C5′), 141.88 (C3′), 138.26 (C3 + C5), 134.31 (C1), 133.16 (C4), 124.72 (C2 + C6), 109.58 (C4′), 47.00 (C4 ${}^{‴}$ ), 42.94 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 31.66 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 21.18 (2 × CH₃), 13.66 (CH₃), 13.41 (CH₃). IR (KBr, cm⁻¹): 3287, 1634, 1536, 1483. HRMS for (C₂₁H₂₆N₈O [M + H]⁺). Calcd: 407.2308. Found: 407.2302.

4.2.2.3

4.2.2.3 3-Chloro-N-(1-(6-(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetrazin-3-yl)piperidin-4-yl)-5-fluorobenzamide (1c)

Yield, 34%. Red solid, mp 194–196 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.64 (s, 1H, H4), 7.49 (d, J = 8.8 Hz, 1H, H2), 7.22 (d, J = 8.0 Hz, 1H, H6), 7.12 (d, J = 7.7 Hz, 1H, NH), 6.12 (s, 1H, H4′), 4.95 (d, J = 13.7 Hz, 2H, N-CH₂-pip), 4.50–4.40 (m, 1H, N-CH-pip), 3.37 (t, J = 12.2 Hz, 2H, N-CH₂-pip), 2.61 (s, 3H, CH₃), 2.32–2.20 (m, 5H, CH₃ + -CH₂-pip), 1.78–1.67 (m, 2H, -CH₂-pip). ¹³C NMR (101 MHz, CDCl₃) δ 164.63 (d, J_CF = 2.5 Hz, C⚌O), 162.42 (d, J_CF = 251.3 Hz, C5), 160.19 (C4″), 156.62 (C1″), 152.03 (C5′), 142.02 (C3′), 137.69 (d, J_CF = 7.4 Hz, C3), 135.28 (d, J_CF = 10.1 Hz, C1), 123.36 (d, J_CF = 3.1 Hz, C4), 118.95 (d, J_CF = 24.9 Hz, C6), 113.10 (d, J_CF = 23.0 Hz, C2), 109.70 (C4′), 47.44 (C4 ${}^{‴}$ ), 42.97 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 31.35 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 13.52 (CH₃), 13.44 (CH₃). ¹⁹F NMR (188 MHz, CDCl₃) δ −109.22. IR (KBr, cm⁻¹): 3261, 1638, 1537, 1484. HRMS for (C₁₉H₂₀ClFN₈O [M + H]⁺). Calcd: 431.1511. Found: 431.1505.

4.2.2.4

4.2.2.4 N-(1-(6-(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetrazin-3-yl)piperidin-4-yl)-2-nitro-4-(trifluoromethyl)benzamide (1d)

Yield, 55%. Red solid, mp 230–231 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.26 (s, 1H, H3), 7.85 (d, J = 7.8 Hz, 1H, H5), 7.63 (d, J = 7.9 Hz, 1H, H6), 6.54 (d, J = 6.5 Hz, 1H, NH), 6.00 (s, 1H, H4′), 4.84 (d, J = 13.7 Hz, 2H, N-CH₂-pip), 4.44–4.31 (m, 1H, N-CH-pip), 3.36 (t, J = 12.1 Hz, 2H, N-CH₂-pip), 2.49 (s, 3H, CH₃), 2.33–2.18 (m, 5H, CH₃ + -CH₂-pip), 1.73–1.51 (m, 2H, -CH₂-pip). ¹³C NMR (101 MHz, CDCl₃) δ 164.77 (C⚌O), 160.32 (C4″), 156.65 (C1″), 152.03 (C5′), 146.34 (C2), 141.96 (C3′), 135.88 (C1), 131.45 (C5), 131.42 (C3), 130.39 (C6), 129.84 (CF₃), 121.93 (q, J_CF = 7.8 Hz, C4), 109.63 (C4′), 47.55 (C4 ${}^{‴}$ ), 42.81 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 31.09 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 13.63 (CH₃), 13.41 (CH₃). ¹⁹F NMR (188 MHz, CDCl₃) δ −63.11. IR (KBr, cm⁻¹): 3267, 1646, 1543, 1326. HRMS for (C₂₀H₂₀F₃N₉O₃ [M + H]⁺). Calcd: 492.1719. Found: 492.1717.

4.2.2.5

4.2.2.5 N-(1-(6-(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetrazin-3-yl)piperidin-4-yl)-3-fluoro-4-methoxybenzamide (1e)

Yield, 46%. Red Solid, mp 229–230 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.50 (d, J = 10.1 Hz, 2H, H2 + H6), 6.88 (t, J = 8.0 Hz, 1H, H5), 6.45 (d, J = 7.0 Hz, 1H, NH), 6.03 (s, 1H, H4′), 4.84 (d, J = 13.2 Hz, 2H, N-CH₂-pip), 4.32 (br, 1H, N-CH-pip), 3.86 (s, 3H, OCH₃), 3.26 (t, J = 12.3 Hz, 2H, N-CH₂-pip), 2.50 (s, 1H, CH₃), 2.31–2.03 (m, 5H, CH₃ + -CH₂-pip), 1.56 (d, J = 10.2 Hz, 2H, -CH₂-pip). ¹³C NMR (101 MHz, CDCl₃) δ 165.49 (C⚌O), 160.19 (C4″), 156.63 (C1″), 152.01 (C5′), 151.74 (d, J_CF = 247.0 Hz, C3), 150.41 (d, J_CF = 10.7 Hz, C4), 141.91 (C3′), 127.07 (d, J_CF = 5.5 Hz, C1), 123.54 (d, J_CF = 3.3 Hz, C6), 115.14 (d, J_CF = 19.7 Hz, C2), 112.56 (d, J_CF = 1.5 Hz, C5), 109.59 (C4′), 56.23 (OCH₃), 47.17 (C4 ${}^{‴}$ ), 42.95 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 31.57 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 13.62 (CH₃), 13.38 (CH₃). ¹⁹F NMR (188 MHz, CDCl₃) δ -133.85.IR (KBr, cm⁻¹): 3261, 1631, 1541, 1485. HRMS for (C₂₀H₂₃FN₈O₂ [M + H]⁺). Calcd: 427.2006. Found: 427.2003.

4.2.2.6

4.2.2.6 4-Cyano-N-(1-(6-(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetrazin-3-yl)piperidin-4-yl)benzamide (1f)

Yield, 30%. Red solid, mp 187–189 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.88 (d, J = 8.1 Hz, 2H, H2 + H6), 7.65 (d, J = 8.0 Hz, 2H, H3 + H5), 6.78 (d, J = 7.6 Hz, 1H, NH), 6.04 (s, 1H, H4′), 4.86 (d, J = 13.7 Hz, 2H, N-CH₂-pip), 4.44–4.30 (m, 1H, N-CH-pip), 3.28 (t, J = 12.2 Hz, 2H, N-CH₂-pip), 2.52 (s, 3H, CH₃), 2.21–2.19 (m, 5H, CH₃ + -CH₂-pip), 1.65–1.54 (m, 2H, -CH₂-pip). ¹³C NMR (101 MHz, CDCl₃) δ 165.26 (C⚌O), 160.22 (C4″), 156.68 (C1″), 152.09 (C5′), 142.01 (C3′), 138.32 (C1), 132.31 (C3 + C5), 127.89 (C2 + C6), 118.02 (CN), 115.03 (C4), 109.75, 47.49 (C4 ${}^{‴}$ ), 43.01 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 31.43 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 13.56 (CH₃), 13.48 (CH₃). IR (KBr, cm⁻¹): 3261, 1636, 1534, 1485. HRMS for (C₂₀H₂₂N₉O [M + H]⁺). Calcd: 404.1947. Found: 404.1940.

4.2.2.7

4.2.2.7 N-(1-(6-(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetrazin-3-yl)piperidin-4-yl)-2-methylbenzamide (1g)

Yield, 36%. Red solid, mp 204–206 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.37–7.30 (m, 2H, H4 + H6), 7.25–7.19 (m, 2H, H3 + H5), 6.12 (s, 1H, H4′), 6.00 (d, J = 7.7 Hz, 1H, NH), 4.92 (d, J = 13.8 Hz, 2H, N-CH₂-pip), 4.46–4.30 (m, 1H, N-CH-pip), 3.45–3.32 (m, 2H, N-CH₂-pip), 2.59 (s, 3H, CH₃), 2.47 (s, 3H, CH₃), 2.37 (s, 3H, CH₃), 2.33–2.24 (m, 2H, -CH₂-pip), 1.67–1.57 (m, 2H, -CH₂-pip). ¹³C NMR (101 MHz, CDCl₃) δ 169.61 (C⚌O), 160.28 (C4″), 156.71 (C1″), 152.05 (C5′), 141.88 (C3′), 136.19 (C1), 135.89 (C2), 131.01 (C4), 129.97 (C3), 126.60 (C6), 125.74 (C5), 109.62 (C4′), 46.88 (C4 ${}^{‴}$ ), 42.86 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 31.63 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 19.71 (CH₃), 13.70 (CH₃), 13.45 (CH₃). IR (KBr, cm⁻¹): 3277, 1629, 1550, 1485. HRMS for (C₂₀H₂₄N₈O [M + H]⁺). Calcd: 393.2151. Found: 393.2146.

4.2.2.8

4.2.2.8 2-Bromo-N-(1-(6-(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetrazin-3-yl)piperidin-4-yl)benzamide (1h)

Yield, 35%. Red solid, mp 134–136 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.45 (d, J = 7.9 Hz, 1H, H6), 7.39 (dd, J = 7.6, 1.5 Hz, 1H, H3), 7.23 (t, J = 7.4 Hz, 1H, H4), 7.15 (td, J = 7.7, 1.6 Hz, 1H, H5), 6.20 (d, J = 7.8 Hz, 1H, NH), 5.98 (s, 1H, H4′), 4.74 (d, J = 13.8 Hz, 2H, N-CH₂-pip), 4.34–4.21 (m, 1H, N-CH-pip), 3.36–3.24 (m, 2H, N-CH₂-pip), 2.45 (s, 3H, CH₃), 2.23 (s, 3H, CH₃), 2.16 (dd, J = 13.0, 2.8 Hz, 2H, -CH₂-pip), 1.63–1.48 (m, 2H, -CH₂-pip). ¹³C NMR (101 MHz, CDCl₃) δ 167.11 (C⚌O), 160.28 (C4″), 156.67 (C1″), 152.02 (C5′), 141.86 (C3′), 137.60 (C1), 133.27 (C3), 131.30 (C5), 129.48 (C4), 127.56 (C6), 119.20 (C2), 109.61 (C4′), 47.19 (C4 ${}^{‴}$ ), 42.72 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 31.32 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 13.71 (CH₃), 13.45 (CH₃). IR (KBr, cm⁻¹): 3253, 1646, 1544. HRMS for (C₁₉H₂₁BrN₈O [M + H]⁺). Calcd: 457.1100. Found: 457.1097.

4.2.2.9

4.2.2.9 N-{1-[6-(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetrazin-3-yl]piperidin-4-yl}-2-naphthamide (1i)

Yield, 66%. Red solid, mp 204–206 °C.¹H NMR (400 MHz, CDCl₃) δ 8.25 (s, 1H, H2), 7.82–7.78 (m, 4H, H3, H6, H7, H8), 7.51–7.46 (m, 2H, H4 + H5), 6.49 (br, 1H, NH), 6.04 (s, 1H, H4′), 4.85 (d, J = 12.1 Hz, 2H, N-CH₂-pip), 4.39 (br, 1H, N-CH-pip), 3.27 (t, J = 12.4 Hz, 2H, N-CH₂-pip), 2.51 (s, 3H, CH₃), 2.28 (s, 3H, CH₃), 2.23 (d, J = 11.3 Hz, 2H, -CH₂-pip), 1.61 (d, J = 9.6 Hz, 2H, -CH₂-pip). ¹³C NMR (101 MHz, CDCl₃) δ 167.12 (C⚌O), 160.26 (C4″), 156.70 (C1″), 152.06 (C5′), 141.91 (C3′), 134.76 (C1), 132.56 (C2a), 131.57 (C6a), 128.87 (C3), 128.46 (C5), 127.75 (C2 + C7), 127.47 (C6), 126.81 (C4), 123.58 (C8), 109.63 (C4′), 47.22 (C4 ${}^{‴}$ ), 42.96 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 29.70 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 13.70 (CH₃), 13.45 (CH₃). IR (KBr, cm⁻¹): 3265, 1637, 1543, 1490, 1236. HRMS (for C₂₃H₂₅N₈O [M + H]⁺). Calcd: 429.2151. Found: 429.2141.

4.2.2.10

4.2.2.10 3-Chloro-N-(1-(6-(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetrazin-3-yl)piperidin-4-yl)benzamide (1j)

Yield, 69%. Red solid, mp 179–181 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.70 (s, 1H, H2), 7.58 (d, J = 7.8 Hz, 1H, H4), 7.35 (d, J = 8.1 Hz, 1H, H6), 7.23 (t, J = 8.0 Hz, 1H, H5), 6.76 (d, J = 7.6 Hz, 1H, NH), 5.99 (s, 1H, H4′), 4.80 (d, J = 13.8 Hz, 2H, N-CH₂-pip), 4.36–4.26 (m, 1H, N-CH-pip), 3.30–3.17 (m, 2H, N-CH₂-pip), 2.47 (s, 3H, CH₃), 2.19 (s, 3H, CH₃), 2.14 (dd, J = 13.0, 2.8 Hz, 2H, -CH₂-pip), 1.56 (qd, J = 12.3, 4.1 Hz, 2H, -CH₂-pip). ¹³C NMR (101 MHz, CDCl₃) δ 165.80 (C⚌O), 160.20 (C4″), 156.62 (C1″), 152.01 (C5′), 141.94 (C3′), 136.20 (C1), 134.54 (C3), 131.45 (C4), 129.78 (C5), 127.43 (C6), 125.23 (C2), 109.65 (C4′), 47.29 (C4 ${}^{‴}$ ), 42.94 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 31.45 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 13.59 (CH₃), 13.44 (CH₃). IR (KBr, cm⁻¹): 3255, 1639, 1540, 1484. HRMS for (C₁₉H₂₂ClN₈O [M + H]⁺). Calcd: 413.1605. Found: 413.1488.

4.2.2.11

4.2.2.11 4-Chloro-N-(1-(6-(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetrazin-3-yl)piperidin-4-yl)-3-sulfamoylbenzamide (1k)

Yield, 65%. Red solid, mp 194–196 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.18 (d, J = 7.3 Hz, 1H, H6), 7.75 (d, J = 3.6 Hz, 1H, H4), 7.53 (d, J = 4.9 Hz, 1H, H3), 7.07 (t, J = 4.3 Hz, 1H, NH), 6.13 (s, 1H, H4′), 4.84 (d, J = 13.6 Hz, 2H, N-CH₂-pip), 4.30–4.23 (m, 1H, N-CH-pip), 3.37 (t, J = 11.8 Hz, 2H, N-CH₂-pip), 2.52 (s, 3H, CH₃), 2.31 (s, 3H, CH₃), 2.13 (d, J = 12.8 Hz, 2H, -CH₂-pip), 1.74 (d, J = 14.1 Hz, 2H, -CH₂-pip). ¹³C NMR (101 MHz, DMSO-d₆) δ 161.39 (C⚌O), 160.31 (C4″), 156.54 (C1″), 151.29 (C5′), 141.73 (C3 + C3′), 140.21 (C4), 130.15 (C1 + C2), 128.26 (C5), 127.54 (C6), 109.31 (C4′), 46.87 (C4 ${}^{‴}$ ), 42.97 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 31.19 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 13.74 (CH₃), 13.17 (CH₃). IR (KBr, cm⁻¹): 2954, 1375, 1541, 1488, 1241. HRMS for (C₁₉H₂₃ClN₉O₃S [M + H]⁺). Calcd: 492.1333. Found: 492.1791.

4.2.2.12

4.2.2.12 4-(Chloromethyl)-N-(1-(6-(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetrazin-3-yl)piperidin-4-yl)benzamide (1l)

Yield, 43%. Red solid, mp 201–203 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.79 (d, J = 8.2 Hz, 2H, H2 + H6), 7.47 (d, J = 8.2 Hz, 2H, H3 + H5), 6.22 (d, J = 7.7 Hz, 1H, NH), 6.12 (s, 1H, H4′), 4.94 (d, J = 13.8 Hz, 1H, N-CH₂-pip), 4.62 (s, 2H, CH₂), 4.45–4.38 (m, 1H, N-CH-pip), 3.42–3.30 (m, 2H, N-CH₂-pip), 2.58 (s, 3H, CH₃), 2.35 (s, 3H, CH₃), 2.28 (dd, J = 12.7, 2.6 Hz, 2H, -CH₂-pip), 1.63 (qd, J = 12.3, 4.2 Hz, 2H, -CH₂-pip). ¹³C NMR (101 MHz, CDCl₃) δ 166.42 (C⚌O), 160.30 (C4″), 156.73 (C1″), 152.10 (C5′), 141.94 (C3′), 141.05 (C4), 134.27 (C1), 128.76 (C3 + C5), 127.40 (C2 + C6), 109.64 (C4′), 47.20 (C—Cl), 45.32 (C4 ${}^{‴}$ ), 42.95 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 31.72 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 13.69 (CH₃), 13.45 (CH₃). IR (KBr, cm⁻¹): 3279, 1645, 1541, 1485, 1238. HRMS for (C₂₀H₂₃ClN₈O [M + H]⁺). Calcd: 427.1756. Found: 427.4261.

4.2.3

4.2.3 General synthetic procedure of compounds 2a-j

A mixture of 3,6-bis(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetrazine 6 (1.0 mmol), TEA (triethylamine) (3 mmol), and the respective arylpiperazine or arylpiperidine (1.5 mmol) in acetonitrile (5 mL) was stirred overnight at room temperature. Then, the mixture was evaporated under vacuum to dryness, and the products were separated by flash column chromatography on silica gel eluting with CH₂Cl₂/MeOH (15:1).

Close

Export to PPT

4.2.3.1

4.2.3.1 3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(4-phenylpiperazin-1-yl)-1,2,4,5-tetrazine (2a)

Yield, 80%. Red solid, mp 196–197 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.32–7.16 (m, 2H, H3 and H5), 6.87 (m, 3H, 3H, H2, H4 and H6), 6.10 (s, 1H, H4′), 4.25–4.10 (m, 4H, 2 × CH₂-pip), 3.40–3.28 (m, 4H, 2 × CH₂-pip), 2.58 (s, 3H, CH₃), 2.35 (s, 3H, CH₃). ¹³C NMR (50 MHz, CDCl₃) δ 160.33 (C4″), 156.79 (C1″), 152.01 (C5′), 150.77 (C1), 141.81 (C3′), 129.22 (C3 + C5), 120.66 (C4), 116.70 (C2 + C6), 109.57 (C4′), 49.11 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 43.55 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 13.64 (CH₃), 13.39 (CH₃). IR (KBr, cm⁻¹): 2921, 2820, 1611, 1561, 1240. HRMS for (C₁₇H₂₀N₈ [M + H]⁺). Calcd: 337.1884. Found: 337.1894.

4.2.3.2

4.2.3.2 3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(4-(4-nitrophenyl)piperazin-1-yl)-1,2,4,5-tetrazine (2b)

Yield, 65%. Red solid, mp 199–201 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.17 (d, J = 8.7 Hz, 2H, H3 + H5), 6.89 (d, J = 8.7 Hz, 2H, H2 + H6), 6.11 (s, 1H, H4′), 4.30–4.08 (m, 4H, 2 × CH₂-pip), 3.72–3.50 (m, 4H, 2 × CH₂-pip), 2.58 (s, 3H, CH₃), 2.35 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 160.38 (C4″), 157.11 (C1″), 154.30 (C5′), 152.37 (C1), 142.01 (C3′), 139.25 (C4), 125.98 (C3 + C5), 113.08 (C2 + C6), 109.91 (C4′), 46.54 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 43.00 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 13.71 (CH₃), 13.55 (CH₃). IR (KBr, cm⁻¹): 2983, 2850, 1597, 1484, 1321, 1240. HRMS for (C₁₇H₁₉N₉O₂ [M + H]⁺). Calcd: 382.1734. Found: 382.1750.

4.2.3.3

4.2.3.3 3-(4-(4-Chlorophenyl)piperazin-1-yl)-6-(3,5-dimethyl-1H-pyrazol-1-yl)-1,2,4,5-tetrazine (2c)

Yield, 69%. Red solid, mp 186–188 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.21 (d, J = 8.9 Hz, 2H, H3 + H5), 6.87 (d, J = 8.9 Hz, 2H, H2 + H6), 6.08 (s, 1H, H4′), 4.24–4.08 (m, 4H, 2 × CH₂-pip), 3.37–3.20 (m, 4H, 2 × CH₂-pip), 2.54 (s, 3H, CH₃), 2.32 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 160.28 (C4″), 156.79 (C1″), 152.01 (C5′), 149.34 (C1), 141.78 (C3′), 129.03 (C3 + C5), 125.45 (C4), 117.83 (C2 + C6), 109.60 (C4′), 49.00 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 43.36 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 13.61 (CH₃), 13.38 (CH₃). IR (KBr, cm⁻¹): 2960, 2820, 1553, 1280, 1240. HRMS for (C₁₇H₁₉ClN₈ [M + H]⁺). Calcd: 371.1494. Found: 371.1508.

4.2.3.4

4.2.3.4 3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(4-(4-methoxyphenyl)piperazin-1-yl)-1,2,4,5-tetrazine (2d)

Yield, 71%. Red solid, mp 147–149 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.01–6.90 (m, 2H, H3 + H5), 6.91–6.79 (m, 2H, H2 + H6), 6.08 (s, 1H, H4′), 4.24–4.11 (m, 4H, 2 × CH₂-pip), 3.76 (s, 3H, OCH₃), 3.26–3.12 (m, 4H, 2 × CH₂-pip), 2.55 (s, 3H, CH₃), 2.33 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 160.30 (C4″), 156.74 (C1″), 154.47 (C4), 151.96 (C5′), 145.03 (C1), 141.78 (C3′), 118.98 (C2 + C6), 114.47 (C3 + C5), 109.53 (C4′), 55.43 (OCH₃), 50.62 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 43.69 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 13.62 (CH₃), 13.37 (CH₃). IR (KBr, cm⁻¹): 2910, 2820, 1513, 1240. HRMS for (C₁₈H₂₂N₈O [M + H]⁺). Calcd: 367.1989. Found: 367.2003.

4.2.3.5

4.2.3.5 3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(4-phenylpiperidin-1-yl)-1,2,4,5-tetrazine (2e)

Yield, 84%. Red solid, mp 143–145 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.39–7.14 (m, 5H, H2, H3, H4, H5, H6), 6.08 (s, 1H, H4′), 3.31–3.12 (m, 2H, CH₂-pip), 3.00–2.80 (m, 2H, CH₂-pip), 2.56 (s, 3H, CH₃), 2.34 (s, 3H, CH₃), 2.01–2.09 (m, 1H, CH), 1.95–1.64 (m, 4H, 2 × CH₂). ¹³C NMR (101 MHz, CDCl₃) δ 160.23 (C4″), 156.55 (C1″), 151.87 (C5′), 144.89 (C1), 141.76 (C3′), 128.60 (C3 + C5), 126.67 (C2 + C6), 126.59 (C4), 109.40 (C4′), 44.44 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 42.54 (C4 ${}^{‴}$ ), 32.75 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 13.67 (CH₃), 13.35 (CH₃). IR (KBr, cm⁻¹): 2923, 2853, 1577, 1486, 1247. HRMS for (C₁₈H₂₁N₇ [M + 2H]²⁺). Calcd: 337.1931. Found: 337.1894.

4.2.3.6

4.2.3.6 3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(4-(pyridin-2-yl)piperazin-1-yl)-1,2,4,5-tetrazine (2f)

Yield, 65%. Red solid, mp 158–160 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.20 (d, J = 4.7 Hz, 1H, H3), 7.51 (t, J = 7.8 Hz, 1H, H5), 6.68 (m, 2H, H4 + H6), 6.07 (s, 1H, H4′), 4.24–4.04 (m, 4H, 2 × CH₂-pip), 3.85–3.67 (m, 4H, 2 × CH₂-pip), 2.54 (s, 3H, CH₃), 2.32 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 160.38 (C4″), 158.80 (C1), 156.78 (C1″), 151.99 (C5′), 147.91 (C3), 141.81 (C3′), 137.68 (C5), 113.97 (C4), 109.57 (C4′), 107.21 (C6), 44.66 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 43.26 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 13.63 (CH₃), 13.38 (CH₃). IR (KBr, cm⁻¹): 2920, 2850, 1490, 1238. HRMS for (C₁₆H₁₉N₉ [M + H]⁺). Calcd: 338.1836. Found: 338.1844.

4.2.3.7

4.2.3.7 3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(4-(pyridin-4-yl)piperazin-1-yl)-1,2,4,5-tetrazine (2g)

Yield, 60%. Red solid, mp 183–185 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.33 (d, J = 5.9 Hz, 2H, H3 + H5), 6.76 (d, J = 6.1 Hz, 2H, H2 + H6), 6.11 (s, 1H, H4′), 4.27–4.12 (m, 4H, 2 × CH₂-pip), 3.67–3.55 (m, 4H, 2 × CH₂-pip), 2.57 (s, 3H, CH₃), 2.35 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 160.38 (C4″), 157.10 (C1″), 154.83 (C1), 152.36 (C5′), 149.12 (C3 + C5), 142.01 (C3′), 109.90 (C4′), 108.43 (C2 + C6), 45.45 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 42.91 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 13.71 (CH₃), 13.56 (CH₃). IR (KBr, cm⁻¹): 2924, 2854, 1611, 1550, 1241. ¹HRMS for (C₁₆H₁₉N₉ [M + H]⁺). Calcd: 338.1836. Found: 338.1847.

4.2.3.8

4.2.3.8 3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)-1,2,4,5-tetrazine (2h)

Yield, 60%. Red solid, mp 183–185 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.43 (s, 1H, H6), 7.68 (d, J = 8.9 Hz, 1H, H4), 6.71 (d, J = 8.8 Hz, 1H, H3), 6.09 (s, 1H, H4′), 4.26–4.04 (m, 4H, 2 × CH₂-pip), 3.96–3.80 (m, 4H, 2 × CH₂-pip), 2.56 (s, 3H, CH₃), 2.56 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 160.44 (C4″), 159.92 (C1), 156.96 (C1″), 152.20 (C5′), 145.74 (q, J_CF = 4.2 Hz, C3), 141.92 (C3′), 134.77 (q, J_CF = 2.9 Hz, C5), 124.37 (q, J_CF = 270.4 Hz, CF₃), 116.00 (q, J_CF = 33.1 Hz, C4), 109.75 (C4′), 105.72 (C6), 44.05 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 43.15 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 13.67 (CH₃), 13.47 (CH₃). ¹⁹F NMR (376 MHz, CDCl₃) δ −61.24. IR (KBr, cm⁻¹): 2923, 2854, 1611, 1277, 1241, 1082, 804. HRMS for (C₁₇H₁₈F₃N₉ [M + H]⁺). Calcd: 406.1710. Found: 406.1720.

4.2.3.9

4.2.3.9 3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(4-(pyrazin-2-yl)piperazin-1-yl)-1,2,4,5-tetrazine (2i)

Yield, 59%. Red solid, mp 139–140 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.20 (d, 1H, J = 1.4 Hz, H6), 8.10 (dd, 1H, J = 2.5, 1.6 Hz, H5), 7.92 (d, 1H, J = 2.6 Hz, H3), 6.09 (s, 1H, H4′), 4.16 (dd, J = 6.4, 4.2 Hz, 4H, 2 × CH₂-pip), 3.81 (dd, J = 6.4, 4.3 Hz, 4H, 2 × CH₂-pip), 2.56 (s, 3H, CH₃), 2.33 (s, 3H, CH₃). ¹³C NMR (50 MHz, CDCl₃) δ 160.45 (C4″), 156.95 (C1″), 154.54 (C1), 152.19 (C5′), 141.92 (C3′), 141.77 (C3), 133.78 (C4), 131.01 (C6), 109.74 (C4′), 43.94 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 43.12 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 13.66 (CH₃), 13.47 (CH₃). IR (KBr, cm⁻¹): 2922, 2853, 1488. HRMS for (C₁₅H₁₈N₁₀ [M + H]⁺). Calcd: 339.1789. Found: 339.1890.

4.2.3.10

4.2.3.10 3-(3,5-Dimethyl-1H-pyrazol-1-yl)-6-(4-(5-nitropyridin-2-yl)piperazin-1-yl)-1,2,4,5-tetrazine (2j)

Yield, 52%. Red solid, mp 201–203 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.21 (d, 1H, J = 1.1 Hz, H6), 8.17–8.08 (m, 1H, H4), 7.93 (d, J = 2.6 Hz, 1H, H3), 6.09 (s, 1H, H4′), 4.26–4.09 (m, 4H, 2 × CH₂-pip), 3.92–3.75 (m, 4H, 2 × CH₂-pip), 2.56 (s, 3H, CH₃), 2.34 (s, 3H, CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 160.46 (C4″), 156.96 (C1″), 154.55 (C1), 152.22 (C5′), 144.39 (C4), 141.93 (C3′), 133.90 (C3), 133.80 (C5), 131.04 (C6), 109.76 (C4′), 43.96 (C3 ${}^{‴}$  + C5 ${}^{‴}$ ), 43.13 (C2 ${}^{‴}$  + C6 ${}^{‴}$ ), 13.68 (CH₃), 13.49 (CH₃). IR (KBr, cm⁻¹): 2922, 2850, 1595, 1329, 1257, 1244. HRMS for (C₁₆H₁₈N₁₀O₂ [M + H]⁺). Calcd: 383.1687. Found: 383.1698.