Modification of Boc-Protected CAN508 via Acylation and Suzuki-Miyaura Coupling

The cyclin-dependent kinase inhibitor, CAN508, was protected with di-tert-butyl dicarbonate to access the amino-benzoylated pyrazoles. The bromo derivatives were further arylated by Suzuki-Miyaura coupling using the XPhos Pd G2 pre-catalyst. The coupling reaction provided generally the para-substituted benzoylpyrazoles in the higher yields than the meta-substituted ones. The Boc groups were only utilized as directing functionalities for the benzoylation step and were hydrolyzed under conditions of Suzuki-Miyaura coupling, which allowed for elimination of the additional deprotection step.

Protecting groups are often used as auxiliary tools in the synthesis of biological active compounds [9].For example, the Boc protection of the endocyclic pyrazole nitrogen was used in the synthesis of TBK1 inhibitor Tozasertib [10], selective CDK4 inhibitors based on the pyrazol-3-yl urea scaffold [11], CDK2 inhibitor PHA-533533 [12], or selective SGK1 inhibitors [13].
The regioselective protection of pyrazoles is inherently complicated due to tautomerism of a pyrazole heterocyclic system [14].Moreover, if a pyrazole system is in conjugation with peripheral functional groups, the tautomeric system is more complex, which results in even more difficult regioselective protection.
The Boc-protection of a 3(5)-aminopyrazole system represents an illustrative example, where both endocyclic nitrogens and the amino groups can be protected concurrently [15,16].The protection usually led to a mixture of mono-and di-substituted regio-isomers, which are difficult to purify.To avoid that, several methods were developed: (i) the acylation of aminopyrazoles with 2 equiv. of acyl reagents followed with saponification of the acyl group attached at the endocyclic pyrazole nitrogen [17]; (ii) the oxidation-reduction sequence of the amino group including Boc-protection of the endocyclic pyrazole nitrogen [18]; and (iii) the direct Boc-protection under alkaline conditions [16].
Furthermore, since the X-ray crystallography of the CAN508-CDK2 complex indicated a possible extension of structure optimization to improve the CDK inhibition activity [4], we initiated a concept to use pyrazole 3 as a starting intermediate for synthetic Route A to give directly unprotected pyrazoles 12 (Scheme 1).
Route A can be potentially an alternative to conventional Route B and offer other synthetic strategies in drug development of pyrazole derivatives with an unprotected endocyclic nitrogen.The synthetic sequence (Route A) employing acylation before the Suzuki-Miyaura coupling (SMC) [19] exposes the catalyst in the SMC reaction to more functionalized reactants/products with a potentially higher coordination activity.
The classical catalysts with triphenylphosphine ligands are not robust and are frequently inhibited or decomposed, when such unprotected functionalized pyrazoles are used.The sterically-bulky phosphines were also inefficient [20,21].Consequently, the SMC reaction has to precede the acylation (Route B) or the pyrazole endocyclic nitrogen is substituted to enable a coupling reaction with reasonable yields [22].Scheme 1. Possible synthetic routes leading to pyrazoles 12.
In the last decade, the development of ligands and their use as pre-catalysts significantly broaden the scope of the SMC coupling with various nitrogen-containing heterocyclic systems [23][24][25].Recently, we have taken an advantage of these scope extensions and reported studies with the challenging aminopyrazole substrates [20,21,26], which encourage us to attempt to synthesize pyrazoles 12 via Route A.
In our endeavours to find more efficient CDK inhibitors based on the structure of pyrazole 1 (CAN508) [14,[27][28][29], we decided to use the Boc-protection of pyrazole 1, subsequent benzoylation of the Boc-protected pyrazole 3, and the final arylation under SMC conditions to further explore the robustness of a catalyst originated from the XPhos Pd G2 pre-catalyst.

Boc-Protection and Benzoylation
We found that the reaction of pyrazole 1 with acyl chlorides proceeded at the endocyclic nitrogen [1].To redirect the acylation reaction toward the amino group it is necessary to protect the endocyclic pyrazole nitrogen and the hydroxyl group.Preliminary experiments with di-tert-butyl dicarbonate provided mono-protected pyrazole 5 and di-protected derivative 3, and showed a significant role of a base (Scheme 2, Route A).While the protection with di-tert-butyl dicarbonate in anhydrous dimethylformamide (DMF) gave pure mono-Boc protected derivative 5, a reaction in pyridine resulted in a mixture of mono-and di-Boc protected derivatives 5 and 3. Another equivalent of di-tert-butyl dicarbonate provided solely pyrazole 3.
To verify that the first mono-Boc protection took place at the diaminopyrazole moiety, we chose an alternative indirect synthetic approach involving an additional protecting group (Scheme 2, Route B).At first, hydrazone 4 was protected with a triisopropylsilyl (TIPS) group to give compound 6, which subsequently underwent cyclyzation with hydrazine to yield pyrazole 7.Then, In the last decade, the development of ligands and their use as pre-catalysts significantly broaden the scope of the SMC coupling with various nitrogen-containing heterocyclic systems [23][24][25].Recently, we have taken an advantage of these scope extensions and reported studies with the challenging aminopyrazole substrates [20,21,26], which encourage us to attempt to synthesize pyrazoles 12 via Route A.
In our endeavours to find more efficient CDK inhibitors based on the structure of pyrazole 1 (CAN508) [14,[27][28][29], we decided to use the Boc-protection of pyrazole 1, subsequent benzoylation of the Boc-protected pyrazole 3, and the final arylation under SMC conditions to further explore the robustness of a catalyst originated from the XPhos Pd G2 pre-catalyst.

Boc-Protection and Benzoylation
We found that the reaction of pyrazole 1 with acyl chlorides proceeded at the endocyclic nitrogen [1].To redirect the acylation reaction toward the amino group it is necessary to protect the endocyclic pyrazole nitrogen and the hydroxyl group.Preliminary experiments with di-tert-butyl dicarbonate provided mono-protected pyrazole 5 and di-protected derivative 3, and showed a significant role of a base (Scheme 2, Route A).While the protection with di-tert-butyl dicarbonate in anhydrous dimethylformamide (DMF) gave pure mono-Boc protected derivative 5, a reaction in pyridine resulted in a mixture of mono-and di-Boc protected derivatives 5 and 3. Another equivalent of di-tert-butyl dicarbonate provided solely pyrazole 3.
To verify that the first mono-Boc protection took place at the diaminopyrazole moiety, we chose an alternative indirect synthetic approach involving an additional protecting group (Scheme 2, Route B).At first, hydrazone 4 was protected with a triisopropylsilyl (TIPS) group to give compound 6, which subsequently underwent cyclyzation with hydrazine to yield pyrazole 7.Then, TIPS-protected pyrazole 7 was treated with di-tert-butyl dicarbonate to obtain derivative 8.The final selective unmasking of the hydroxyl group with TBAF resulted in the target pyrazole 5.While the alternative synthesis of pyrazole 5 confirmed the position of the Boc group at the diaminopyrazole moiety, the single crystal X-ray analysis of 5 unequivocally determined the exact position of the Boc group at the pyrazole endocyclic nitrogen (Figure 2).X-ray crystallography also confirmed the position of the second Boc group at the hydroxyl in pyrazole 3. Selected bond lengths and angles of 3 and 5 can be found in the Supplementary file.Moreover, the X-ray analysis also revealed that the crystal structures of  While the alternative synthesis of pyrazole 5 confirmed the position of the Boc group at the diaminopyrazole moiety, the single crystal X-ray analysis of 5 unequivocally determined the exact position of the Boc group at the pyrazole endocyclic nitrogen (Figure 2).X-ray crystallography also confirmed the position of the second Boc group at the hydroxyl in pyrazole 3. Selected bond lengths and angles of 3 and 5 can be found in the Supplementary file.Moreover, the X-ray analysis also revealed that the crystal structures of  To confirm the markedly preferred reactivity of the hydroxyl over the amino groups, we carried out benzoylation of pyrazole 5 with benzoyl chloride (Scheme 3).This reaction resulted in pyrazol 9. Subsequent deprotection with diluted trifluoroacetic acid (TFA) provided benzoylated pyrazol 10.The predominant reactivity of the hydroxyl group highlighted the necessity of its protection.The acylation of pyrazole 3 with benzoyl chlorides provided amino-benzoylated pyrazoles 11ac (Table 1).Since benzoylated pyrazoles 11a and 11b were accompanied with an increased amount of impurities, the crude products were directly deprotected in the next step without isolation.The subsequent cleavage of both Boc groups was carried out initially under conventional conditions with diluted TFA to yield 12a (Table 1, entry 1).In view of the following Suzuki-Miyaura coupling, tripotassium phosphate was additionally used to deprotect pyrazoles 11b-c in the very good to excellent yields (Table 1, entries 2 and 3).To confirm the markedly preferred reactivity of the hydroxyl over the amino groups, we carried out benzoylation of pyrazole 5 with benzoyl chloride (Scheme 3).This reaction resulted in pyrazol 9. Subsequent deprotection with diluted trifluoroacetic acid (TFA) provided benzoylated pyrazol 10.The predominant reactivity of the hydroxyl group highlighted the necessity of its protection.To confirm the markedly preferred reactivity of the hydroxyl over the amino groups, we carried out benzoylation of pyrazole 5 with benzoyl chloride (Scheme 3).This reaction resulted in pyrazol 9. Subsequent deprotection with diluted trifluoroacetic acid (TFA) provided benzoylated pyrazol 10.The predominant reactivity of the hydroxyl group highlighted the necessity of its protection.The acylation of pyrazole 3 with benzoyl chlorides provided amino-benzoylated pyrazoles 11ac (Table 1).Since benzoylated pyrazoles 11a and 11b were accompanied with an increased amount of impurities, the crude products were directly deprotected in the next step without isolation.The subsequent cleavage of both Boc groups was carried out initially under conventional conditions with diluted TFA to yield 12a (Table 1, entry 1).In view of the following Suzuki-Miyaura coupling, tripotassium phosphate was additionally used to deprotect pyrazoles 11b-c in the very good to excellent yields (Table 1, entries 2 and 3).The acylation of pyrazole 3 with benzoyl chlorides provided amino-benzoylated pyrazoles 11a-c (Table 1).Since benzoylated pyrazoles 11a and 11b were accompanied with an increased amount of impurities, the crude products were directly deprotected in the next step without isolation.The subsequent cleavage of both Boc groups was carried out initially under conventional conditions with diluted TFA to yield 12a (Table 1, entry 1).In view of the following Suzuki-Miyaura coupling, tripotassium phosphate was additionally used to deprotect pyrazoles 11b-c in the very good to excellent yields (Table 1, entries 2 and 3).

Suzuki-Miyaura Coupling
In order to extend the diversity of pharmacologically relevant amino benzoylated pyrazoles 12b-c, we used our previously reported SMC conditions for dinitropyrazoles [26].Preliminary deprotection experiments with tripotassium phosphate showed that already two thirds of 11c were converted after 30 min into 12c and after 1 h the cleavage was almost complete.The first coupling experiments with p-tolylboronic acid enabled for providing biphenyl 12d in a very good yield (Table 2, entry 1), which indicated the robustness of the catalyst [30].The comparable yields were obtained with benzene and 4-methoxybenzeneboronic acids (entries 2 and 3).The ortho-substitution did not affect the reactivity negatively; it was possible to isolate 12g in the excellent yield (entry 4).However, the electron-withdrawing nitro group decreased the yield (entry 5).Electron-rich furan-3-yl and thiophen-3-yl boronic acids reacted with 11c in the very good or excellent yields (entries 6 and 7).Thiophen-3-yl 96 1 1 Reagents and conditions: XPhos Pd G2 (5 mol %), K3PO4 (4 equiv.),(hetero)arylboronic acid (2 equiv.),1,4-dioxane/water 4:1, 100 °C, 24 h. 2 Isolated yield. 3 After 12 h, the same amount of 4-nitroboronic acid and XPhos Pd G2 was added and the reaction was proceeded for the next 24 h.

Suzuki-Miyaura Coupling
In order to extend the diversity of pharmacologically relevant amino benzoylated pyrazoles 12b-c, we used our previously reported SMC conditions for dinitropyrazoles [26].Preliminary deprotection experiments with tripotassium phosphate showed that already two thirds of 11c were converted after 30 min into 12c and after 1 h the cleavage was almost complete.The first coupling experiments with p-tolylboronic acid enabled for providing biphenyl 12d in a very good yield (Table 2, entry 1), which indicated the robustness of the catalyst [30].The comparable yields were obtained with benzene and 4-methoxybenzeneboronic acids (entries 2 and 3).The ortho-substitution did not affect the reactivity negatively; it was possible to isolate 12g in the excellent yield (entry 4).However, the electron-withdrawing nitro group decreased the yield (entry 5).Electron-rich furan-3-yl and thiophen-3-yl boronic acids reacted with 11c in the very good or excellent yields (entries 6 and 7).

Suzuki-Miyaura Coupling
In order to extend the diversity of pharmacologically relevant amino benzoylated pyrazoles 12b-c, we used our previously reported SMC conditions for dinitropyrazoles [26].Preliminary deprotection experiments with tripotassium phosphate showed that already two thirds of 11c were converted after 30 min into 12c and after 1 h the cleavage was almost complete.The first coupling experiments with p-tolylboronic acid enabled for providing biphenyl 12d in a very good yield (Table 2, entry 1), which indicated the robustness of the catalyst [30].The comparable yields were obtained with benzene and 4-methoxybenzeneboronic acids (entries 2 and 3).The ortho-substitution did not affect the reactivity negatively; it was possible to isolate 12g in the excellent yield (entry 4).However, the electron-withdrawing nitro group decreased the yield (entry 5).Electron-rich furan-3-yl and thiophen-3-yl boronic acids reacted with 11c in the very good or excellent yields (entries 6 and 7).Since the benzoylation of pyrazole 11b was accompanied by the mono-Boc impurity, and it was found that the catalyst is already exposed to a substantial excess of unprotected pyrazoles 12b and 12c in the earliest stage of the coupling, we decided to perform the coupling reaction directly with unprotected pyrazole 12b.Preliminary attempts showed a more difficult coupling reaction at the meta position.The HPLC analyses usually confirmed the presence of unreacted pyrazole 12b, the desired coupling product, and other unknown impurities.The yields were considerably lower and only pyrazoles 12k-n were possible to isolate (Table 3).Since the benzoylation of pyrazole 11b was accompanied by the mono-Boc impurity, and it was found that the catalyst is already exposed to a substantial excess of unprotected pyrazoles 12b and 12c in the earliest stage of the coupling, we decided to perform the coupling reaction directly with unprotected pyrazole 12b.Preliminary attempts showed a more difficult coupling reaction at the meta position.The HPLC analyses usually confirmed the presence of unreacted pyrazole 12b, the desired coupling product, and other unknown impurities.The yields were considerably lower and only pyrazoles 12k-n were possible to isolate (Table 3).Thiophen-3-yl 60 1 Reagents and conditions: XPhos Pd G2 (5 mol %), K3PO4 (4 equiv.),(hetero)arylboronic acid (2 equiv.),1,4-dioxane/water 4:1, 100 °C, 24 h. 2 Isolated yield.

General
All starting materials are commercially available.Commercial reagents were used without purification.Melting points were determined on a Boetius stage and are uncorrected.Flash column chromatography was performed on silica gel (pore size 60 Å, 40-63 mm particle size).The LC/MS analyses were carried out on a UHPLC-MS system consisting of UHPLC chromatography Accela with photodiode array detector and triple quadrupole mass spectrometer TSQ Quantum Access (both Thermo Scientific, Santa Clara, CA, USA), using Nucleodur Gravity C18 column at 30 °C and flow rate of 800 μL/min (Kinetex, Phenomenex, 2.6 μm, 2.1 × 50 mm, Torrance, CA, USA).The mobile phase was (A) 0.01M ammonium acetate in water, and (B) acetonitrile, linearly programmed from 10% to 80% B over 2.5 min, kept for 1.5 min.The column was reequilibrated with a 10% of solution B for 1 min.The atmospheric-pressure chemical ionization (APCI) source operated at discharge current of 5 μA, vaporizer temperature of 400 °C, and capillary temperature of 200 °C.The HRMS analyses were carried out on HRMSdExactive (Orbitrap) MS, Thermo Scientific, Santa Clara, CA, USA.The 1 H-and 13 C-NMR spectra were measured in DMSO-d6 on a Bruker Avance 300 FT NMR spectrometer (Billerica, MA, USA) and on a Varian 400 MHz FT NMR spectrometer (Palo Alto, CA, USA).The single crystal X-ray data of 3 (CCDC 1453321) and 5 (CCDC 1453320) were obtained using an Xcalibur2 diffractometer equipped with a Sapphire2 CCD detector (Oxford Diffraction Ltd., Abingdon, UK), and with MoKα radiation (monochromator Enhance, Oxford Diffraction Ltd) and ω-scan technique at 120 K.Additional details regarding structure determinations, such as crystal data and structure refinements, selected bond lengths and angles of covalent as well as non-covalent contacts are summarized in the Supplementary File.

General
All starting materials are commercially available.Commercial reagents were used without purification.Melting points were determined on a Boetius stage and are uncorrected.Flash column chromatography was performed on silica gel (pore size 60 Å, 40-63 mm particle size).The LC/MS analyses were carried out on a UHPLC-MS system consisting of UHPLC chromatography Accela with photodiode array detector and triple quadrupole mass spectrometer TSQ Quantum Access (both Thermo Scientific, Santa Clara, CA, USA), using Nucleodur Gravity C18 column at 30 • C and flow rate of 800 µL/min (Kinetex, Phenomenex, 2.6 µm, 2.1 × 50 mm, Torrance, CA, USA).The mobile phase was (A) 0.01M ammonium acetate in water, and (B) acetonitrile, linearly programmed from 10% to 80% B over 2.5 min, kept for 1.5 min.The column was reequilibrated with a 10% of solution B for 1 min.The atmospheric-pressure chemical ionization (APCI) source operated at discharge current of 5 µA, vaporizer temperature of 400 • C, and capillary temperature of 200 • C. The HRMS analyses were carried out on HRMSdExactive (Orbitrap) MS, Thermo Scientific, Santa Clara, CA, USA.The 1 Hand 13 C-NMR spectra were measured in DMSO-d 6 on a Bruker Avance 300 FT NMR spectrometer (Billerica, MA, USA) and on a Varian 400 MHz FT NMR spectrometer (Palo Alto, CA, USA).The single crystal X-ray data of 3 (CCDC 1453321) and 5 (CCDC 1453320) were obtained using an Xcalibur2 diffractometer equipped with a Sapphire2 CCD detector (Oxford Diffraction Ltd., Abingdon, UK), and with MoKα radiation (monochromator Enhance, Oxford Diffraction Ltd) and ω-scan technique at 120 K.Additional details regarding structure determinations, such as crystal data and structure refinements, selected bond lengths and angles of covalent as well as non-covalent contacts are summarized in the Supplementary File.
reaction mixture was allowed to stir at room temperature for 18 h.After that the solvent was evaporated under reduced pressure and the residue was diluted in methanol (5 mL).The solution was added dropwise to ice water (25 mL).The precipitate was filtered-off, washed with water, and dried in the air to give 5. Yield 122 mg (91%) as a yellow solid; m.p. 114-116 • C; (4-((Triisopropylsilyl)oxy)phenyl)carbonohydrazonoyl dicyanide (6): Hydrazone 4 (186 mg, 1.0 mmol) [1,4] was dissolved in a mixture of dry DCM (5 mL) and DMF (0.5 mL), then imidazole (75 mg, 1.1 mmol) and triisopropylsilyl chloride (TIPS-Cl; 235 µL, 1.1 mmol) were added under continuous stirring and cooling on an ice-bath.The reaction mixture was stirred at room temperature for 18 h.After that, the solvent was removed under reduced pressure and the residue was diluted in methanol (2 mL).The solution was added dropwise to ice water (10 mL).The precipitate was filtered-off, washed with water, and dried in the air.Crude hydrazone 6 was crystallized from methanol (3 mL) to give a yellow solid.Yield 202 mg (59%); m.p. 136-138 • C;

3 Figure 1 .
Figure 1.Derivatives of pyrazole 1 protected at the endocyclic pyrazole nitrogen and hydroxyl group.

Figure 1 .
Figure 1.Derivatives of pyrazole 1 protected at the endocyclic pyrazole nitrogen and hydroxyl group.
both compounds are stabilized by hydrogen bonds of the N-H•••N, N-H•••O and O-H•••N types, situating the individual molecules into supramolecular 1D-polymeric chains (see Figures S1 and S2 in the Supplementary File).Each of the supramolecular chains is connected through a variety of the non-covalent contacts (e.g., N-H•••N, C-H•••O and/or C-H•••N) with two neighboring ones, thus forming a supramolecular-layered structure (see Figures S3 and S4 in the Supplementary File).
both compounds are stabilized by hydrogen bonds of the N-H•••N, N-H•••O and O-H•••N types, situating the individual molecules into supramolecular 1D-polymeric chains (see Figures S1 and S2 in the Supplementary File).Each of the supramolecular chains is connected through a variety of the non-covalent contacts (e.g., N-H•••N, C-H•••O and/or C-H•••N) with two neighboring ones, thus forming a supramolecular-layered structure (see Figures S3 and S4 in the Supplementary File).

Molecules 2018, 23, 149 4 of 14 TIPS
-protected pyrazole 7 was treated with di-tert-butyl dicarbonate to obtain derivative 8.The final selective unmasking of the hydroxyl group with TBAF resulted in the target pyrazole