Synthesis of pyridyl substituted pyrazolo[4,3-c ]pyridines as potential inhibitors of protein kinases

A synthetic route towards 3-(2-pyridyl)-6-(hetero)aryl-1 H -pyrazolo[4,3-c ]pyridines is described. The key step consists of a microwave-assisted multi-component reaction, including a Sonogashira type cross-coupling of appropriate 5-chloropyrazole-4-carbaldehydes with alkynyl-(hetero)arenes followed by pyridine ring formation of the coupling products in the presence of tert -butylamine, directly affording the title compounds. A congener without substituent at N-1 was accessed via cleavage of a tert -butyl protecting group. Detailed NMR spectroscopic studies ( 1 H, 13 C and 15 N) were undertaken with the obtained compounds. Selected representatives were evaluated for their potential as inhibitors of protein kinases.


Introduction
2][3] Thus, in the last years considerable effort has been devoted to the development of small molecule inhibitors of CDKs as potential drug candidates for oncology. 1,2,46][7] Naturally, the latter compound has inspired further exploration of new CDK inhibitors by variation of the substituents and by repositioning of nitrogen atoms on the purine scaffold.Thus, various congeners containing 2-5 nitrogen atoms in the bicyclic core have been considered.In the course of these investigations, representatives of four classes of bioisosteres revealed improved biological properties, namely pyrazolo [4,3-d]pyrimidines (A), pyrazolo [1,5-a]pyrimidines (B), pyrazolo [1,5-a]pyridines (C) and pyrazolo [1,5-a] [1,3,5]triazines (D) (Figure 1). 8,9In view of this we became interested in somewhat related compounds of type E, characterized by a pyrazolo [4,3-c]pyridine core and carrying at least one pyridyl substituent in order to provide a slightly basic side-chain (Figure 1).

Synthetic study
Recently, we have presented a novel and general access to the synthesis of pyrazolo [4,3-c]pyridines via Sonogashira cross-coupling of 5-chloropyrazole-4-carbaldehydes and subsequent pyridine ring closure of the resulting 5-alkynylpyrazole-4-carbaldehydes in the presence of tertbutylamine as the nitrogen source. 10According to this approach, the envisaged synthetic pathway to the target compounds of type 6 and 7, respectively, is presented in Scheme 1. β-Keto ester 2, obtained via condensation of 2-acetylpyridine (1) with diethyl carbonate, should be transformed into the appropriate pyrazolone 3 and the latter by Vilsmeier reaction -with concomitant transformation of the OH group into a chloro functionality -into the corresponding N-protected 5-chloropyrazole-4-carbaldehyde 4. Subsequently, Sonogashira coupling reaction with terminal alkynes should generate the respective alkyne intermediate 5, which in the presence of tertbutylamine should undergo ring closure into the respective pyrazolo[4,3-c]pyridine 6. Optional removal of the protecting R 1 should provide congeners of type 7 unsubstituted at pyrazole N-1.

Scheme 1.
Envisaged synthetic route to the target compounds 6 and 7.
2][13] Unfortunately, an orienting Vilsmeier-Haak formylation 14 applied to 2-(4-methoxybenzyl)-2,4-dihydro-3H-pyrazol-3-one demonstrated that the PMB group does not survive the harsh conditions of this reaction.For this reason, the more stable but harder to cleave benzyl group and the even less vulnerable methyl group were considered as possible R 1 protecting groups.The synthesis of pyrazolones 3 (3a: R 1 = Me, 3b.R 1 = PhCH2) was accomplished via reaction of 3-oxoester 2the latter obtained by condensation of 2-acetylpyridine (1) with diethyl carbonatewith methylhydrazine and benzylhydrazine, respectively.The high electron density at position 4 of the pyrazole system of compounds 3a,b permits smooth Vilsmeier-Haack formylation (DMF/POCl3), whereas due to the presence of excessive POCl3 the OH-group (the tautomeric equivalent of pyrazolone C=O) is simultaneously transformed into a chloro function to afford 5-chloropyrazole-4-carbaldehydes 4a,b in good yields. 15In recent studies we have shown that 1-substituted 5-chloropyrazole-4-carbaldehydes are suitable reactants in Sonogashira cross-coupling reactions with appropriate alkynes. 10,16lthough the general order of reactivity for substrates is known to be R-I > R-Br ~ R-OTf > R-Cl, 17 the activated chloro atom in the latter turned out to be a good leaving group.Surprisingly, aldehydes 4a,b proved to be fairly inert in cross-couplings with phenylacetylene or 2-ethynylpyridine under standard conditions.Neither did variation of the Pd catalyst and/or solvent, nor applying the reaction under microwave assistance, lead to satisfying results, i.e. to the formation of coupling products 5 in acceptable yields.However, a breakthrough was achieved when Sonogashira coupling and subsequent pyridine ring closure to bicycles 6 were carried out as a one-pot multi-component reaction.Thus, microwave heating (800 W, 150 °C, 1h) of aldehydes 4 with an alkyne and excessive tert-butylamine in DMF in the presence of PdCl2(PPh3)2 afforded pyrazolopyridines 6a-d in acceptable to good yields (Scheme 2).It should be emphasized that in recent years such multi-component reactions (MCRs) have attracted considerable attention due to their unmatched synthetic efficiency which permits the construction of complex molecules in an elegant and sufficient manner. 18,19heme 2. Synthesis of pyrazolo [4,3-c]pyridines 6a-f and 7a.
In order to prepare N-unsubstituted congeners, for instance 7a, which could be used in biological tests, removal of the N-1 protecting group in the corresponding compounds 6 was necessary.It is known from the literature that N-methyl groups can be removed from N-1 of pyrazoles or condensed pyrazoles by melting the N-methyl derivatives with anhydrous pyridine hydrochloride. 20However, when compounds 6a-c were subjected to such reaction conditions (210 °C) in most cases complex reaction mixtures resulting from decomposition reactions were obtained.2][23] Either unreacted starting material was obtained or decomposition was observed.As benzylic protecting groups alternatively can be removed by oxidative methods, also the system KOt-Bu/DMSO 24 was applied, but no reaction was observed.Furthermore, treatment with TFA, HBr or AlCl3 in toluene was not successful.As an alternative, the tert-butyl protecting group was considered; this has been successfully employed in pyrazole chemistry. 25Thus, pyrazolone 3c (R 1 = tert-butyl) was prepared from 2 and tert-butylhydrazine and subsequently converted into aldehyde 4c (R 1 = tert-butyl, Scheme 1), however in low yields.Multi-component reaction with 3-alkynylpyridine or 3-alkynylthiophene, respectively, afforded bicycles 6e and 6f.The deprotection of 6e was tried under different conditions, for instance in TFA, TFA-H2O (rt and afterwards at 120 °C), as well in HCOOH, HCl, at rt and reflux, where no reaction was observed.Finally, the removal of the protecting group was accomplished by treatment of 6e with conc.H2SO4 affording the target compound 7a (Scheme 2).

NMR Spectroscopic investigations
Pyrazolones of type 3 are capable of prototropic tautomerism. 26,27Discounting participation of the pyridine system in the tautomerism, in principle three different tautomeric forms are possible, i.e. the OH (A), the CH (B) and the NH form (C) (Figure 2, upper row).The 1 H and the 13 C NMR spectra of pyrazolones 3a (R 1 = Me), 3b (R 1 = PhCH2) and 3c (R 1 = tert-butyl) clearly show that these compounds are exclusively present as CH-isomers B in CDCl3 solution due to the appearance of a CH2 fragment at position 4 of the pyrazole ring (for instance 3b: 1 H: 3.81 ppm, 2H; corresponding 13 C: 38.2 ppm, CH2 multiplicity according to APT; Figure 2, lower row, left).
Because pyrazolone 3b showed limited solubility in CDCl3, spectra were also taken of a DMSO-d6 solution.In the latter solvent, 3b turned out to exist as 5-hydroxypyrazole (form A), as confirmed by the appearance of pyrazole C-4 as CH-fragment ( 1 H: 5.98 ppm, 13 C: 84.5 ppm), the Due to the low solubility of 7a in chloroform the NMR spectra were recorded of a DMSO-d6 solution.In principle, for 7a annular prototropic tautomerism at the pyrazole ring is possible (proton attached to N-1 or N-2).Moreover, the flexible proton theoretically could be also located at N-5 or at the pyridine N-atom of the 3-substituent, however the latter possibilities can be definitely ruled out considering the 15 N chemical shifts of the concerning nitrogen atoms.Nevertheless, a distinct NOE between the acidic proton (δ 13.89 ppm) and H-7 (δ 8.17 ppm) unequivocally confirms preference for the N1-H form (Figure 3, right).Moreover, the similarity of 1 H, 13 C and 15 N NMR chemical shifts of 7a with those of the 'fixed' N-methyl congener 6c (Figure 3, left) strongly confirms this assignment.Because pyrazolone 3b showed limited solubility in CDCl3, spectra were also taken of a DMSO-d6 solution.In the latter solvent, 3b turned out to exist as 5-hydroxypyrazole (form A), as confirmed by the appearance of pyrazole C-4 as CH-fragment ( 1 H: 5.98 ppm, 13 C: 84.5 ppm), the 13 C chemical shift of pyrazole C-5 (153.2 ppm) and particularly by the 15 N chemical shift of N-2 (−102.2) ppm (Figure 2, lower row, right), the latter definitely ruling out the NH-form C.
Due to the low solubility of 7a in chloroform the NMR spectra were recorded of a DMSO-d6 solution.In principle, for 7a annular prototropic tautomerism at the pyrazole ring is possible (proton attached to N-1 or N-2).Moreover, the flexible proton theoretically could be also located at N-5 or at the pyridine N-atom of the 3-substituent, however the latter possibilities can be definitely ruled out considering the 15 N chemical shifts of the concerning nitrogen atoms.Nevertheless, a distinct NOE between the acidic proton (δ 13.89 ppm) and H-7 (δ 8.17 ppm) unequivocally confirms preference for the N1-H form (Figure 3, right).Moreover, the similarity of 1 H, 13 C and 15 N NMR chemical shifts of 7a with those of the 'fixed' N-methyl congener 6c (Figure 3, left) strongly confirms this assignment.

Biological tests
Our aim was to prepare a new scaffold of protein kinase inhibitors.Therefore, the biological activity of prepared compounds 6 and 7 was assayed as described previously. 28The compounds have been tested in kinase inhibition assays for their inhibitory potency towards recombinant CDK2, CDK9, CK2 and c-Abl, and for their cytotoxicity against various cancer cell lines.None of the tested compounds proved biological activity, but we consider these results to be preliminary and further modification of the molecules are necessary to identify first hits amongst our pyrazolo [4,3-c]pyridines.Most known kinase inhibitors are heterocyclic organic molecules that act like ATP competitors, interacting through one to three hydrogen bonds with the hinge region of the kinase. 1 Our future work will focus on modifications that would create typical Hbond donor-acceptor motif and on modifications increasing the polarity (solubility).

Experimental Section
General.Melting points were determined with a Reichert-Kofler hot-stage microscope.Mass spectra were obtained on a Shimadzu GP 1000 instrument (EI, 70 eV), a Bruker maXis 4G instrument (ESI-TOF, HRMS) and a Finnigan MAT 8230 instrument (EI, 70 eV, HRMS). 1 H, 13 C and 15 N NMR spectra were recorded with a Bruker Avance III 400 spectrometer at 293 K (400 MHz for 1 H, 100 MHz for 13 C, 40 MHz for 15 N).The center of the solvent signal was used as an internal standard which was related to TMS with δ 7.26 ppm ( 1 H in CDCl3), δ 2.49 ppm ( 1 H in DMSO-d6), δ 77.0 ppm ( 13 C in CDCl3), δ 39.5 ppm ( 13 C in DMSO-d6). 15N NMR spectra (gs-HMBC, gs-HSQC) (40.56 MHz) were obtained using a "directly" detecting broadband observe (BBFO) probe were referenced against neat, external nitromethane.Digital resolutions were 0.25 Hz/data point in the 1 H and 0.4 Hz/data point in the 1 H-coupled 13 C-NMR spectra (gated decoupling).Unequivocal assignments of signals was carried out by the combined application of standard NMR spectroscopic techniques such as 1 H coupled 13 C-NMR spectra, APT, HMQC, gs-HSQC, gs-HMBC, COSY, TOSCY, NOESY and NOE difference spectroscopy. 29The elemental analyses were performed at the microanalytical laboratory (Faculty of Chemistry) and were in good agreement (+/-0.4%)with the calculated values.For the microwave reaction system an Anton Paar Synthos 3000 was employed.Light petroleum refers to the fraction with boiling point 40-65 °C.Yields are not optimized.

13 C
chemical shift of pyrazole C-5 (153.2 ppm) and particularly by the 15 N chemical shift of N-2 (−102.2) ppm (Figure 2, lower row, right), the latter definitely ruling out the NH-form C.