Unprecedented synthesis of a 14-membered hexaazamacrocycle

The transformation of 3-[(ethoxymethylene)amino]-1-methyl-1H-pyrazole-4-carbonitrile into the 14-membered macrocycle, 2,10-dimethyl-2,8,10,16-tetrahydrodipyrazolo[3,4-e:3',4'-l][1,2,4,8,9,11]hexaazacyclotetradecine-4,12-diamine, by the reaction with excess hydrazine under various conditions was studied in detail. The reaction proceeded through the initial formation of 4-imino-2-methyl-2,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-amine followed by dimerization to give the final macrocycle. A convenient synthesis of the latter starting from 4-imino-2-methyl-2,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-amine was developed. A plausible pathway for the macrocycle self-assembly is discussed. Some features of the structure and reactivity of the obtained macrocycle are outlined.


Results and Discussion
The readily available 3-amino-1-methyl-1H-pyrazole-4-carbonitrile (3) was used as the starting material.This compound was prepared according to the described regioselective method [42] based on the reaction of malononitrile with triethyl orthoformate followed by subsequent treatment of the obtained dinitrile 2 with benzaldehyde methyl hydrazone in benzene, conc.aqueous HCl in EtOH, and NaOH in water (Scheme 1).The key intermediate of the macrocycle preparation, imidate 4, was synthesized using the reported procedure [43] by refluxing a solution of aminopyrazole 3 in triethyl orthoformate.
First, we studied the reaction of imidate 4 with hydrazine hydrate in EtOH under Dolzhenko's conditions (4 equivalents of N 2 H 4 •H 2 O, concentration of 4 = 0.5 mmol/mL, reflux, 2 h) [40].The resulting precipitate was isolated by filtration and washed with EtOH.In contrast to the reported data, the yield of the obtained product was significantly lower and did not exceed 38%.Moreover, according to NMR spectroscopic data, the isolated product was a mixture of the desired macrocycle 5 and a noticeable amount of an impurity (Scheme 2) whose formation was not mentioned in the cited reference.
The structure of the concomitant impurity was established using 1D and 2D NMR spectroscopy.The 1 H NMR spectrum in DMSO-d 6 shows the presence of two methylpyrazole moieties (singlet signals of two methyl groups at 3.63 and 3.70 ppm, singlet signals of two CH protons of pyrazole rings at 7.81 and 7.84 ppm), a H-N-C-H fragment with trans-orientation of protons (two doublets at 9.87 and 7.50 ppm, 3 J = 11.2Hz), four NH 2 groups at 6.27, 5.72, 5.59, and 4.61 ppm (singlets).Signals of 11 different carbon atoms including 8 carbons of two methylpyrazole moieties were observed in the 13 С NMR spectrum.Thus, we concluded that the impurity has bis-pyrazole structure 6.This structure was also confirmed by the 1 H, 13 C-HSQC and 1 H, 13 C-HMBС spectra, as well as by comparing the experimental carbon chemical shifts in DMSO-d 6 with those calculated for 6 by the GIAO method at the PBE1PBE/6-311+G(2d,p) level of theory using the DFT B3LYP/6-311++G(d,p) optimized geometries (DMSO solution) and applying a multi-standard approach [44] (see the Supporting Information File 1 for details).The high-resolution mass spectrum (ESI + ) of a mixture of 5 and the impurity, in addition to a peak at m/z = 329.1696[M + H] + for compound 5, shows a peak at m/z = 319.1862[M + H] + , consistent with the molecular formula of C 11 H 18 N 12 for bis-pyrazole 6.According to NMR spectroscopic data, the amount of bis-pyrazole 6 in the crude product formed under above conditions was about 18 mol %.
The structure of macrocycle 5 was confirmed by comparing its 1 H and 13 C NMR spectra with those reported in ref. [40].It should be noted that the 1 H and 13 C{1H} NMR spectra of compound 5 in DMSO-d 6 show only a half-number set of proton or carbon signals (five and six signals, respectively), thus indicating its C 2 -symmetric dimeric structure.The analysis of 2D NMR spectroscopic data provided additional evidence for the macrocycle 5 structure (see Supporting Information File 1).The high-resolution mass spectrum (ESI + ) confirmed its chemical formula as C 12 H 16 N 12 .
Thus, we found that, in contrast to the reported data [40], the reaction between imidate 4 and hydrazine hydrate (4 equiv) in refluxing EtOH for 2 h afforded macrocycle 5 in a relatively low yield, along with an appreciable amount of the byproduct 6.This prompted us to optimize the reaction conditions varying hydrazine hydrate excess (from 3.1 to 4.3 equivalents), solvent (EtOH, MeOH, 1,4-dioxane, DME), reaction time (2 h and 6 h), and also using anhydrous hydrazine instead of hydrazine hydrate.However, all our attempts to improve both the yield and the purity of 5 failed.For example, prolonging the reaction time between 4 and N 2 H 4 •H 2 O (4 equiv) in refluxing EtOH to 6 h resulted in an increase in the purity of the macrocycle (5/6 = 91:9), but simultaneously to a decrease in its yield to 25%.Reducing the amount of hydrazine hydrate to 3 equivalents (EtOH, reflux, 2 h) had a similar effect and gave an 85:15 mixture of 5 and 6 in an overall yield of 22%.In refluxing MeOH (3 equiv of N 2 H 4 •H 2 O, 2 h), a mixture of 5 and 6 in a ratio of 89:11 was obtained in 31% overall yield.In aprotic solvents (1,4-dioxane or DME), the selectivity of the reaction dramatically decreased and a mixture of 5 and 6 along with significant amounts of various unidentified byproducts was formed.For example, the reaction of 4 with N 2 H 4 •H 2 O (4.1 equiv) in refluxing 1,4-dioxane for 2 h, followed by evaporation of the volatiles under reduced pressure, afforded a complex mixture containing only 7 mol % of macrocycle 5 according to the 1 H NMR spectrum with the addition of a weighted amount of succinimide as a reference.Analogously, only 2 mol % of 5 were detected under the above conditions (1,4-dioxane, reflux, 2 h) when anhydrous hydrazine (4.2 equiv) was used as a promoter.
A plausible pathway for the transformation of imidate 4 into macrocycle 5 is shown in Scheme 3.This pathway includes fast substitution of the ethoxy group by hydrazine to give the intermediate amidrazone 7 followed by its rapid conversion to pyrazolopyrimidine 8. Slow dimerization of compound 8 results in macrocycle 5.The formation of 5 through pyrazolopyrimidine 8 is confirmed by the literature data [40] that the reaction of 4 with N 2 H 4 •H 2 O in EtOH to give 8 proceeds under much milder conditions than the reaction to afford 5 (rt and reflux, respectively).Based on this background, we assumed that the synthesis of macrocycle 5 could be carried out directly from 8. We also hoped that this would be especially useful from a preparative viewpoint, since pure pyrazolopyrimidine 8 can be easily obtained in any required quantities, in contrast to pure imidate 4.
Pyrazolopyrimidine 8 was prepared by the reaction of 4 with N 2 H 4 •H 2 O in EtOH according to our modification of the described procedure [40] using room temperature (without precooling), a lower excess of N 2 H 4 •H 2 O (1.6 equiv instead of 5 equiv) and a shorter reaction time (1 h instead of 5 h).The precipitated compound 8 was isolated by filtration in a 96% yield (Scheme 4).Previously, the structure of 8 was assigned based on 1 H and 13 C NMR spectroscopic data [40].However, these data are insufficient to distinguish compound 8 and its isomer 9 resulting from a Dimroth rearrangement that is known to proceed in 3-substituted 4-iminopyrimidine systems [40,[45][46][47].Our analysis of 1 H, 13 C NMR, and 2D NMR spectra (DMSO-d 6 solution) of the prepared product confirmed its structure as compound 8.For example, the 1 H, 13 C-HMBC spectrum showed correlation of the NH 2 protons with carbon C-6 (through three bonds), and the 1 H, 1 H-NOESY experiment revealed a diagnostic NOE between the NH 2 and H-6 protons.The structure 8 was also confirmed by comparing the experimental carbon chemical shifts of the prepared compound in DMSO-d 6 with shifts calculated for 8 and 9 by the GIAO method at the PBE1PBE/6-311+G(2d,p) level of theory using the DFT B3LYP/6-311++G(d,p) optimized geometries (DMSO solution) and applying a multi-standard approach [44].The calculated shifts of sp 2 -atoms C-7a, C-4, C-6, C-3, and C-3a in (Z)-8 and the s-cis-conformer (with respect to the C4-N bond) of 9 were 156.6, 152.1, 151,9, 128.3, 105.6 ppm and 160.9, 158.9, 157.4,123.3, 98.4 ppm, respectively.The corresponding experimental shifts (155.0,151.8, 149.9, 128.4,105.4 ppm) were in good agreement with the structure 8.It is noteworthy that the DFT B3LYP/6-311++G(d,p) calculations using the PCM solvation model showed that (Z)-8 was significantly less stable than the s-cisconformer of 9 in DMSO solution (ΔG = 7.17 kcal/mol; 298 K, 1 atm).
The dimerization of 8 was thoroughly studied varying promoter, its amount, solvent, substrate concentration, and reaction time (Table 1).
First, we studied the dimerization of 8 promoted by hydrazine hydrate in EtOH under reflux (Table 1, entries 1-3).We found that the starting material was completely consumed in the presence of 3 equivalents of N 2 H 4 •H 2 O within 2 h and the precipitated solid was isolated by filtration.According to the 1 H NMR spectrum, this crude product was a mixture of macrocycle 5 and bis-pyrazole 6 in a molar ratio of 80:20 (Table 1, entry 2).An increase in the amount of N 2 H 4 •H 2 O to 6.3 equivalents led to a faster conversion of 8, however, the amount of bis-pyrazole 6 in the isolated mixture increased to 36% (Table 1, entry 3).In contrast, reducing the amount of N 2 H 4 •H 2 O to 1 equivalent resulted in an increase in the 5:6 ratio to 89:11 and a decrease in the conversion of 8 to 63% after 2 h of reflux (Table 1, entry 1).
The experimental data described above were obtained using 50-141 mg of pyrazolopyrimidine 8.We demonstrated that, under the optimized conditions (1.5 equiv of N 2 H 4 •H 2 O, MeOH, reflux, 3 h), the reaction can be scaled up to gram quantities without any loss of efficiency and even with a noticeable Under these conditions, the cleavage of either the C2-N3 bond or the N1-C2 bond can proceed.In the latter case, the formed 3-amino-N-(hydrazonomethyl)-1-methyl-1H-pyrazole-4carboximidohydrazide recyclizes to give compound 10.
Thermodynamic parameters for the hydrazine-promoted transformation of pyrazolopyrimidine 8 into macrocycle 5 in MeOH solution were estimated by the DFT B3LYP/6-311++G(d,p)  calculations.Relative Gibbs free energies of the starting, final, and intermediate molecular systems (Figure 1) were calculated using the Gibbs free energies for the most stable isomers of 8, macrocycle 5, intermediates A, C, D, and hydrazine.
Figure 1 shows that the hydrazine-promoted transformation of pyrazolopyrimidine 8 into macrocycle 5 in MeOH is a thermodynamically favorable process.Moreover, the extremely low solubility of macrocycle 5 makes the dimerization of 8 even more preferable.
It  width of the signals decreases, however, even at 100 °С, some signals remain broadened.
The failure in the synthesis of compounds 11 and 12 can be associated with difficulties in the annulation of the triazole rings due to the high conformational rigidity of macrocycle 5 (see Supporting Information File 1).Thus, instead of the formation of 11 and 12, side reactions occur leading to the splitting of the macrocyclic ring in 5.The relatively low stability and tendency to disintegration of the macrocyclic ring in 5 can be explained by its somewhat anti-aromatic character (see above).

Conclusion
In conclusion, self-assembly of 14-membered 1,2,4,8,9,11hexaazamacrocycle annulated with two pyrazole rings, 2,10dimethyl-2,8,10,16-tetrahydrodipyrazolo[3,4-e:3',4'l] [1,2,4,8,9,11]hexaazacyclotetradecine-4,12-diamine (5), proceeding by the reaction of 3-[(ethoxymethylene)amino]-1methyl-1H-pyrazole-4-carbonitrile (4) with excess hydrazine was reinvestigated in detail.Under all tested conditions, including the reported ones, the product yield was significantly lower compared with the reported one and did not exceed 38%.Moreover, in all cases the isolated product was a mixture of the desired macrocycle and a noticeable amount of the admixture of bis-pyrazole structure (>18-19 mol %).We demonstrated that the reaction proceeds through the initial formation of 4-imino-2methyl-2,4-dihydro-5H-pyrazolo [3,4-d]pyrimidin-5-amine (8) undergoing dimerization to give the final macrocycle.Preparative protocol for the macrocycle synthesis on a multi-gram scale starting from 4-imino-2-methyl-2,4-dihydro-5H-pyrazolo [3,4d]pyrimidin-5-amine (8) was developed.Under the optimized conditions (1.5 equiv of N 2 H 4 •H 2 O, MeOH, reflux, 3 h), the macrocycle 5 was obtained in a 35% isolated yield after crystallization.We believe that, despite the moderate yield of the macrocycle, the ease of its isolation and purification, the operational simplicity of all the reactions, the high availability of all the reactants make the developed synthesis very promising.A plausible pathway of the hydrazine-promoted self-assembly of the macrocycle from the pyrazolopyrimidine based on the experimental data and the DFT calculations was proposed.It involves nucleophilic attack of hydrazine on the C2 carbon of the pyrimidine ring followed by cleavage of the C2-N3 bond, dimerization of the bis-amidrazone formed, and macrocyclization of the dimer.The DFT calculations also showed that the hydrazine-promoted transformation of the pyrazolopyrimidine 8 into the macrocycle 5 in MeOH is a thermodynamically favorable process with ΔG = −5.90kcal/mol at 298 K and 1 atm.We found that the 14-membered macrocyclic ring can be destroyed under harsh reaction conditions.Thus, under reflux in CH(OEt) 3 in the presence of HCOOH or in AcOH, the macrocycle afforded pyrazolo[ Since numerous heterocyclic and carbocyclic analogs of 3-amino-1-methyl-1H-pyrazole-4-carbonitrile (3), which is the starting material in the synthesis of macrocycle 5, are readily available, we believe that our results will be useful for the preparation of other polyunsaturated annulated 14-membered 1,2,4,8,9,11-hexaazamacrocycles.

Experimental
All solvents and liquid reagents purchased from commercial sources were distilled prior to use.Petroleum ether had a distillation range of 40-70 °C.100% Hydrazine hydrate was used in the syntheses.Anhydrous N 2 H 4 was obtained from N 2 H 4 •H 2 O according to the standard procedure.All other reagents were purchased from commercial sources and used without additional purification.FTIR spectra were recorded using a Bruker Alpha-T spectrophotometer in KBr.Band characteristics in the IR spectra are defined as very strong (vs), strong (s), medium (m), weak (w), shoulder (sh), and broad (br).NMR spectra (solutions in DMSO-d 6 or CDCl 3 ) were acquired using a Bruker Avance III 600 spectrometer at 600.13 ( 1 H) and 150.90 ( 13 C) MHz. 1 H NMR chemical shifts are referenced to the residual proton signal in DMSO-d 6 (2.50 ppm) or CDCl 3 (7.24ppm).In 13 C NMR spectra, the central signal of DMSO-d 6 (39.50 ppm) or CDCl 3 (77.23 ppm) was used as a reference.Multiplicities are reported as singlet (s), doublet (d), triplet (t), quartet (q), and some combinations of these, multiplet (m).Selective 1 H-1 H decoupling, DEPT-135 experiments as well as HMQC, HMBC, and NOESY correlation techniques were used to aid in the assignment of 1 H and 13 C NMR signals.Elemental analyses (CHN) were performed using a Thermo Finnigan Flash EA1112 apparatus.High-resolution mass spectra (HRMS) were obtained using a Bruker mikrOTOF II focus spectrometer (ESI + ).All yields refer to isolated and spectroscopically pure material.The color of the solids is white if not otherwise mentioned.The geometry optimizations were carried out at the B3LYP level of theory using Gaussian 16 suite [55] of quantum chemical programs.Pople's basis sets, 6-311++G(d,p), was employed for geometry optimization.The effect of continuum solvation was incorporated by using the polarizable continuum model (PCM).
Enthalpies and Gibbs free energies were obtained by adding unscaled zero-point vibrational energy corrections (ZPVE) and thermal contributions to the energies (temperature 298.150Kelvin, pressure 1.000 atm).Carbon chemical shifts of the prepared compounds in DMSO were calculated by the GIAO method at the PBE1PBE/6-311+G(2d,p) level of theory using the DFT B3LYP/6-311++G(d,p) optimized geometries (DMSO solution) and applying a multi-standard approach [44].

3-[(Ethoxymethylene
)amino]-1-methyl-1H-pyrazole-4carbonitrile (4): Imidate 4 was prepared according to the literature method [43].Our modification of the method is provided below in details.A solution of aminopyrazole 3 [42] (9.044 g, 74.10 mmol) in HC(OEt) 3 (145 mL) was stirred under reflux for 23 h.After the reaction had completed (monitored by 1 H NMR spectroscopy), the obtained solution was concentrated under water pump vacuum upon heating in a water bath at 65 °C.The product was extracted from the resulting dense brown oil by trituration 4 times with a mixture of ether (25 mL) and petroleum ether (50 mL), and 1 time with a mixture of ether (10 mL) and petroleum ether (20 mL) at room temperature.The extraction was considered as completed when the brown oil solidified.The product precipitated upon concentration of the combined extracts under reduced pressure.The concentration was carried out until a suspension convenient for filtering was obtained (not to dryness).The suspension was cooled (−18 °C), the precipitate was filtered, washed with cold petroleum ether (3 × 20 mL), and dried to give imidate 4 (10.373g, 79%) as a very light creamy solid which was used in the next step.Method B: Compound 13 (0.034 g, 59%) as a light yellow solid was prepared from macrocycle 5 (0.055 g, 0.17 mmol), СН(ОЕt) 3 (4 mL) and НСООН (0.016 mL) (reflux, 8 h) as described in Method A.

Table 1 :
Synthesis of macrocycle 5 by the dimerization of pyrazolopyrimidine 8. a

Table 1 :
[44]hesis of macrocycle 5 by the dimerization of pyrazolopyrimidine 8. a (continued) according to1H NMR spectrum of the crude product; d calculated based on overall mass yields and molar ratios of the products; e plus a small amount of unidentified impurities; f plus a significant amount of unidentified impurities; g the yield was estimated by1H NMR spectrum for a mixture of the crude product with a weighed amount of succinimide as a reference.increaseinthemacrocycleyieldupto41%(Table1,entries20and 21).The extremely poor solubility of product 5 in most organic solvents allowed to purify it from all admixtures, inaccording to NMR data (Table1, entries 22 and 23).It is noteworthy that triazole 10 was the major product in dioxane (10/5/6 = 86.5:13:0.5,Table1,entry23).Again, macrocycle 5 was separated from this mixture by crystallization from DMF, and pyrazolyl-1,2,4-triazole 10 was isolated from the mother liquor.The structure of compound 10 was established based on 1D and 2D NMR spectroscopic data.The 1 H NMR spectrum in DMSOd 6 showed signals of two amino groups at 6.15 and 5.46 ppm.NOEs were observed between the NH 2 group at 6.15 ppm and two CH protons (8.28 and 8.12 ppm).Additionally, the structure 10 was confirmed by comparing its experimental carbon chemical shifts in DMSO-d 6 with shifts calculated by the GIAO method at the PBE1PBE/6-311+G(2d,p) level of theory using the DFT B3LYP/6-311++G(d,p) optimized geometries (DMSO solution) and applying a multi-standard approach[44].It is noteworthy that, in the most stable conformer of 10, the NH 2 group of the triazole ring is located between the pyrazole and triazole ring protons providing the observed NOEs.withN 2 H 4 •H 2 O (4.0 equiv) in 1,4-dioxane for 2 h followed by removal of the volatiles under reduced pressure to obtain a mixture of 5 and 6 in only a slightly changed ratio (84:16 according to the 1 H NMR spectrum).It should be noted that Scheme 6 also explains formation of pyrazolyl-1,2,4-triazole 10 by the reaction of 8 with N 2 H 4 •H 2 O in the presence of TsOH•H 2 O (Table 1, entries 22 and 23).