Michael Cyclization of Polarized Systems : Synthesis and in vitro Anti-Diabetic Evaluation of Some Novel Pyrimidine , Pyridine , Pyrazole and Pyrazolo [ 3 , 4-b ] pyridine Derivatives

Various interesting heterocycle skeletons were synthesized via Michael type addition reaction with 1,2; 1,3-bidentate nitrogen and carbon nucleophiles. Cycloaddition of different α,β–unsaturated systems afforded bromopyrimidinone 3/5, bromothiazine 4 and bromopyrazole 6a/6b pyrazole-1-carboxylate 8, pyridinylmethanone 9, nicotinonitrile 10, pyrazolopyridine 11a/11b, pyran-3-carbonitrile 12/13, chromenopyridine 14 and N-butyrylpyrazolyl-1-butanone 15 derivatives. The structures of the synthesized compounds were elucidated based on IR, NMR and mass spectral analyses. Group of the newly synthesized compounds were screened for their anti-diabetic activities, whereas compounds 8 and 11b exhibited promising anti-diabetic activities at micro molar concentration against α-glucosidase inhibitor with IC50 values ranging between 13.80-500 μM. On the other hand compound 10 showed a week effect as compared to the standard anti-diabetic agent.


INTRODUCTION
3][4][5][6][7][8][9][10][11] In this respect and various approaches for the preparation of these privileged structures with drug-like properties have been developed on various synthetic strategies.Pyrimidines, as the most important nitrogen-containing heterocyclic compounds, are of chemical and pharmacological interest.Many studies have been shown that compounds containing the pyrimidine ring possess antidiabetic, antibacterial, antifungal, antimalarial, and anticonvulsant activities, [12][13][14][15][16] and anticancer activities, [17,18] and many of pyrimidine compounds were reported to act as calcium channel blockers, [19] and as potential central nervous system (CNS) depressants. [20,21]Also, Pyrazoline derivatives widely occur in the environment, in the form of alkaloids, vitamins and pigments as constituents of plant and animal cell.Considerable attention has been carried out on the pyrazolines and substituted pyrazolines due to their inspiring biological activities such as antibacterial, antifungal, [22][23][24] antidepressant, [25][26][27][28] anticonvulsant, [29] and antitumor, [30] properties.The pyrazoline, a versatile moiety is present as the core constituent in a variety of leading drugs such as Sildenafil, Celebrex, and Rimonabant etc.The fact that α,β-unsaturated carbonyl compounds are push-pull olefins, and Michael acceptors permits them as adaptable intermediates in the synthesis of many azole and azine molecules. [31]Changes in their structure have offered a high degree of diversity that has proven useful for the development of new therapeutic agents having improved potency and lowered toxicity.Based on the above considerations and in continuation of our research on biologically potent heterocyclic derivatives, [32][33][34][35][36][37][38] herein, we report the synthesis, structural elucidation and antidiabetic activity of some new azoles and azines prepared from commercially available reagents.

N EXPERIMENTAL
All melting points were determined using a Stuart melting point apparatus by the open capillary tube method and are uncorrected.IR spectra were recorded on a FT-IR JASCO 6100 instrument in KBr phase. 1 H NMR and 13 C NMR spectra were recorded on a Varian spectrometer (300 MHz) in DMSO-d6 as solvent, using TMS as internal standard and chemical shifts are expressed as δ ppm.The mass spectra were recorded by ISQ LT single quadrupole mass spectrometer.In vitro antimicrobial activities were carried out at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Egypt.Elemental analyses were performed by the Micro Analytical Center, Cairo University, Egypt and are within 0.4 % of the theoretical values.The starting materials 2,3-dibromo- en-1-one (7b) was prepared as described in the literature. [31]The progress of the reaction and the purity of the compounds were routinely monitored on TLC by pre-coated aluminum silica gel 60F254 thin layer plates obtained from Merck (Germany) eluting with petroleum ether/ethyl acetate.The yields of all products were not optimized.All reagents used were obtained from commercial sources.All solvents were of analytical grade and used without further purification.

General Procedure for Synthesis of Nicotinonitrile 10,Pyrazolopyridine 11a and 11b Derivatives
A mixture of 7a/7b (10 mmol) with malononitrile and/or N-phenyl pyrazolone derivative (10 mmol) in acetic acid (30 mL) in presence of ammonium acetate (10 g) was refluxed for 10 h.After removal of the solvent under vacuum, water was added and the resultant solution was neutralized with diluted HCl.The precipitated solid was filtered off, washed with water, dried and recrystallized from the proper to give 10, 11a and 11b, respectively.

α-Glucosidase Inhibitory Assay
The α-glucosidase (Sigma-Aldrich) inhibitory activity was measured according to the method previously described [42] with slight modifications. 1 mg of each sample or acarbose at different concentrations (500 µL) of 1.0 U mL -1 αglucosidase solution in 100 mM phosphate buffer (100 mM, pH 6.8) at 37 °C for 20 minutes.The absorbance of the released p-nitrophenol was measured at 405 nm.The inhibition percentage was calculated using the given formula: Inhibition % = Abs control -Abs sample x 100.

RESULTS AND DISCUSSION
The heterocyclization of 2,3-dibromo-1,3-diarylpropan-1one 1a,b was investigated with the aim of obtaining some better antimicrobial heterocyclic derivatives in a facile route according to the process depicted in scheme 1.The synthetic strategy for the chemical transformation of dibromo ketone to heterocyclic systems depends upon the base induced dehydrobromination forming β-bromo unsaturated ketone conjugated addition of reagent nucleophilic center followed by heterocyclization through the loss of H2O.Thus, Treatment of methanolic solution of 2,3-dibromo-1,3-diphenylpropan-1-one (1a) with 2aminobenzenethiol at room temperature, then heating under reflux afforded the cyclic product 2.The base mediated nucleophilic attack of thiolate anion to the more electrophilic carbon of dibromo ketone 1 followed by thiazine cyclization afforded benzothiazine derivative 2. The IR spectrum of compound 2 revealed the appearance of absorption bands at 3347 (NH) and 1677 (C=O) cm -1 .It's 1 H NMR spectrum also showed a singlet at δ 3.61 ppm assigned to NH and two deshielded doublet of doublet at δ 6.69, 5.78 ppm attributed for (SCHCO) and (Ar-CH) protons as well as mass spectrum showed ion peaks at m / z = 333 (M + + 2), 332 (M + + 1), 331 (M + ) which confirmed their chemical structure.
The [3+3] cyclocondensation of compound 1b with urea in presence of AcONa via one-pot procedure afforded 5-bromo pyrimidin-2(1H)-one derivative 3 through the intermediate A that suffer air oxidation leading to the final product.IR spectrum of the pyrimidinone 3 showed absorption band at 1653 cm -1 assigned to carbonyl group and its 1 H NMR spectrum showed a singlet at δ 8.07 ppm indicated the formation of aminic (NH) and aromatic protons were observed at δ 8.06-7.06ppm that confirmed the structure.Also, mass spectrum of 3 showed molecular ion peak at m / z 392 corresponding to its molecular formula C17H12BrClN2O2.
Although the detailed mechanism of above reaction remains not to be fully clarified, the formation of compound 3 could be explained by a reaction sequence presented in scheme 2. We proposed that the reaction proceeded via a reaction sequence of dehydrobromination, Michael type addition, cyclization and dehydration.Furthermore, the kinetic controlled thiazine derivative 4 was readily obtained through smooth dehydrobromination of the dibromide 1b followed by the S-alkylation of thiourea and subsequent thiazine cyclization via losing H2O (Scheme 1).The postulated mechanism of formation of the thiazine 4 was outlined in details in scheme 3.
Appearance of absorption bands of NH groups in the IR spectrum of compound 4 at 3417 cm -1 and its 1 H NMR spectrum revealed a singlet at δ 3.78 (Ar-CH), a multiplet δ 8.21-7.04(Ar-H + D2O exchangeable NH) as well as the mass spectrum showed ion peaks at m / z 411 (M + + 2) and 410 (M + + 1) confirmed its molecular formula.
Refluxing of ethanolic solution of compound 1b and semicarbazide in presence of AcONa afforded dihydropyrimidin-2(1H)-one derivative 5 (Scheme 1).This was potentiated from spectral analysis which revealed a peaks at 3450 (NH), 3287, 3221 (NH2) 1677, (C=O) cm -1 and the 1 H NMR spectrum showed a singlet at δ 6.60 ppm (D2O exchangeable NH + NH2). 13C NMR spectrum exhibited two signals at δ 55.94, 54.90 corresponding to methylenic CH and methoxy groups.MS showed a peaks at m / z 329, 328 (M + -Br) that confirmed its molecular formula.It seemed that semicarbazide undergo [3 + 3] cycloaddition with the conjugated intermediate formed upon basic dehydrobromination of compound 1b followed by dehydration and isomerization resulting in the formation of dihydropyrimidine derivative 5 and none of pyrazole derivative F was obtained (Scheme 4).
Also, one-pot reaction of compounds 1a,b with hydrazine hydrate (99 %) / AcONa in ethanol under reflux afforded 4-bromopyrazole derivatives 6a and 6b.Absence of absorption bands in the latter series for carbonyl group in the IR spectra as well as in the 1 H NMR spectra was characteristic for the 4-bromo-1H-pyrazoles. Thus, Compound 6a were characterized with absorption band at 3445 cm -1 for (NH) group and absence of the carbonyl in the IR spectrum.Its 1 H NMR spectrum showed signal at δ 13.35 ppm attributed to NH (D2O exchangeable) and the mass spectrum showed ion peaks at m / z 301, 299 and 222 corresponding to (M + + 2), (M + ) and (M + -C6H5), respectively which confirmed its chemical structure.IR spectrum of compound 6b exhibited absorption band at 3280 cm -1 for (NH).A multiplet at δ 7.98-7.10ppm assigned to (Ar-H + D2O exchangeable NH) in the 1 H NMR spectrum and characteristic signals for bromopyrazole showed in 13 C NMR spectrum.Also the mass spectrum showed molecular ion peak at m / z 363 and (M + + 2) at 365 that confirmed its molecular formula C16H12BrClN2O.The formation of compounds 6a and 6b could be explained by a reaction sequence presented in scheme 5.
We proposed that the bromopyrazole derivatives 6a/6b were obtained as the result of dehydrobromination, Michael type addition and subsequent intramolecular cyclodehydration.
Using the behavior of α,β-unsaturated carbonyl system 7 which seemed to be of suitable located functionality towards some nitrogen and/or active methylene nucleophilic reagents was investigated.The mechanistic pathway for these heterocyclization depends upon 1,4-addition followed by intramolecular cyclodehydration and air oxidation in some cases.Thus, when chalcone 7a was allowed to react with tert-Butyl carbazate, cyclization occurred smoothly by heating under reflux to afford pyrazole 8 as shown in scheme 6.It presumably that the pyrazole derivative 8 was obtained via [3 + 2] intermolecular cycloaddition through the αnitrogen of the nucleophilic reagent were added exclusively to α,β-unsaturated system with simultaneous ring closure.Hence, the structure of compound 8 was characterized with absorption bands at 3426 (NH), 1707 (C=O) cm -1 in IR spectrum and the presence of characteristic singlet at δ 10.81, 2.18 and 1.46 ppm in 1 H NMR spectrum for NH, methinyl CH and tert-butyl protons, respectively.
The keeping of α,β-unsaturated carbonyl derivative 7a with ammonium acetate/AcOH under reflux resulted in pyridine cyclization via the nonisolable β-amino-ketone followed by [3 + 3] cyclodehydration to furnish the dihydropyridine derivative, which in turn underwent dehydrogenation giving the final product 9 (Scheme 6).The analytical and spectral data were consistent with the proposed structure.Thus, the IR spectrum of 9 revealed a peak at 1658 cm -1 of the carbonyl group and the 1 H NMR spectrum showed a multiplet at δ 8.27-7.09indicating the Ar-H and CH pyridine protons and a singlet at δ 3.83 ppm according to 2OCH3 protons.
The high yield of α,β-unsaturated system of type 7 encouraged us again to study their further reactivities towards cyano methylene reagents.Pyridine of type 10 was formed upon the addition of malononitrile to ketonic derivative 7 in the presence of ammonia source.Thus, malononitrile added its nucleophilic carbon to electrophilic carbon of 7a producing acyclic Michael type adduct that cyclizes intramolecularly in presence of AcONH4/AcOH producing pyridin-3-carbonitrile 10.While, α,β-unsaturated system 7a,b when allowed to react with 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one afforded pyrazolo [3,4-b]pyridine of type 11 (Scheme 6).The analytical and spectral data of the obtained products were in agreement with the assigned structures.Thus, the 1 H NMR spectrum of 10 showed beside the expected signals of the pyridine moiety, a singlet at δ 3.36 ppm corresponding to D2O exchangable, NH2 group, a multiplet at δ 8.12-6.93ppm including the aromatic protons with CH pyridine and the IR spectrum exhibited peak at 2217 cm -1 of the cyano group.
Also, the 1 H NMR spectrum of product 11a (as an example) showed beside the expected signals of the pyrazolopyridine moiety, a multiplet at δ 8.27-7.09ppm including the aromatic protons with CH pyridine and the IR spectrum exhibited peak at 1600 (C=N) cm -1 and absence of carbonyl groups.The mass spectrum of product 11a revealed molecular ion peak at m / z = 426 that confirmed the postulated structure.
Cycloaddition of cyanoacetamide with the chalcone 7b in ethanolic EtONa solution afforded an Michael type adducts intermediate which in turn underwent basic cyclization followed by dehydrogenation producing pyran-3carbonitrile derivative 13 (Scheme 7).The analytical and spectral data was consistent with the proposed structure.Thus, the IR spectrum of compound 13 showed absorption bands at 3409, 2226, 1771, 1614 cm -1 for OH, CN, C=O and C=C groups, respectively.Its 1 H NMR spectrum showed signal at 10.10 ppm assigned for OH group as well as in the MS showed ion peak at m / z 324 (M + + 1), and 323 (M + ) confirmed its molecular formula.Also, chromenopyridine 14 was prepared by nucleophilic reaction of malonamide with 7b forming the nonisolable phenol A that undergo NH3 losing by intramolecular cyclization to give pyran ring (Scheme 8).Structure of 14 assigned by IR, 1 H NMR and mass spectral data.In its IR spectrum, there are four bands assignable to NH, 2CO, C=C groups at 3433, 1777, 1638 and 1616 cm -1 , respectively. 1 H NMR spectrum showed deshielded singlet at 12.42 ppm attributed for NH (D2O exchangable) and aromatic protons were observed at δ 8.50-7.36ppm.

Biological Activity
14][15][16] From the structure-activity relationships, it is revealed that the selected group of heterocyclic skeletons is important for anti-diabetic studies, which supports the previous results.For instance, the novel compounds 5, 8, 10, 11a and 11b shared similar chemical features and functional groups, such as the presence of hydroxyl and methoxy groups were evaluated for their potential α-glucosidase inhibitory activity.The results are presented in Table 1.The activity comparison and the structure correlation of the tested group of the novel compounds had shown that these potencies paralleled the pyrazole moiety.The pyrazole derivative 8 of the tested compounds were found to have less inhibitory (α-glucosidase inhibitory) activity than a commercial anti-hyperglycemic drug, acarbose (IC50 = 12.87 µM).The pyrazolo [3,4-b]pyridin-4-yl]phenol derivative 11b was the most potent inhibitor (IC50 = 13.80 µM).Also, It was noted that the introduction of a 2-hydroxyphenyl group at position 4 on the condensed pyrazolopyridine moiety increased the activity (compound 11b), while the introduction of a 2methoxyphenyl group at position 4 on the condensed pyrazolopyridine moiety decreased the activity (compound 11a), except for isolated pyrazole (Compound 8).These results indicate that hydroxylation of the condensed pyrazolopyridine rings is important for α-glucosidase inhibitory activity.
The IC50 value was defined as the concentration of alpha-glucosidase inhibitor to inhibit 50 % of its activity under the assayed conditions.All determinations were carried out in triplicate manner and values are expressed as the mean ± CD.Anti-diabetic activity of the tested compounds was depicted with IC50 value in Table 1.

CONCLUSIONS
In this work some novel condensed pyrimidine, pyrazole and pyridine derivatives were synthesized and assayed for their anti-diabetic inhibitory.The experimental work involves the synthesis of α,β-unsaturated carbonyls and α,β-dibromocarbonyls which was then heteroannelated with various 1,2; 1,3-bidentate nitrogen and carbon nucleophiles.Correlations are useful because they can indicate a predictive relationship that can be exploited in practice.Comparing of the anti-diabetic activity of the tested group of new compounds and its analogous described in the literature, [39][40][41] it is obvious that the highest activity might be attributed to the presence of pyrazolo [3,4-b]pyridine moiety bearing 2-HOC6H4 group.