Synthesis and cytotoxic activity of new pyrazolo[1,5-a ]pyrimidines and determination of pyrimidine regiospecific ring formation with 2D NMR

Novel pyrazolo[1,5-a ]pyrimidines (9a, 9b , and 10a-c) were synthesized in high and efficient yields. Their pathway involves the formation of N,S-ketene derivatives (7 a and 7b) that reacted with hydrazine hydrate to get the key intermediate aminopyrazolo derivatives ( 8a and 8b) . These aminopyrazoles were further reacted with either acetylacetoneor β-ketoesters resulting in the targeted pyrazolopyrimidines (9a, 9b , and 10a-c) . All prepared compounds were fully characterized by spectral methods and the cyclization of 10a-d was proved by 2D NMR such as HMBC, HSQC and NOESY. The targeted pyrazolopyrimidines were subjected to in vitro anticancer screening and all of them showed promising cytotoxic activity when compared to doxorubicin. Compound 10d was the most active with IC 50 = 1.98, 2.20 and 2.61µM against MCF-7 , BT474 and A549 cancer cell lines.


Introduction
The development of new anticancer agents was a major area of research during the last decade.Instead of that complete over control of cancer has not been achieved yet. 1 Beside to surgery, chemotherapy is still one of the most important medical options.3][4] In addition, pyrazolo-pyrimidine base scaffolds are belonging to a biologically active class as they are structurally related to intrinsic purine bases.6][7] Compound 2 was reported to be an effective anti-tumour agent and a scaffold to adenosine (ATP) binding receptor in several kinases. 6Moreover, various synthetic methods of pyrazolopyrimidines have been described, [7][8][9][10][11] and one of these methods involves the formation of ketene-S,S or N,S-acetals.3][14][15] are famous with their potency that enable them to be effective anti-cancer agents.Based on these findings, and as a continuation of previous work, [16][17][18][19][20] we synthesized new expected drug hybrid of two active moieties pyrazolopyrimidine and benzothiazole or oxazole.The main objective of the research is to synthesize two series (9a,b) and (10a-d) in a convenient method as a new combination of pyrazolpyrimidine based scaffold substituted with benzothiazole or oxazole.This method starts by converting of either 4-aminophenyl-benzothiazole or oxazole to their corresponding 5aminopyrazole derivatives as a common precursor to pyrazolopyrimidines when reacted with diketones or β-ketoesters.These series of hybrids might be of more synergistic activity against MCF-7, BT474 and A549 cancer cell lines than the initial entities.Also, one of research objectives is directed toward exploring the mechanism of pyrazolopyrimidine cyclization through reaction of 5-aminopyrazole and β-ketoesters.

Results and Discussion
The reaction of amines 5a or 5b 14 with ethyl cyanoacetate in DMF gave 2-cyano-N-substituted acetamido derivatives 6a and 6b.The structure of 6a was confirmed by its 1 H NMR that showed (CH2C≡N) at δ3.97.6a and 6b were further reacted with phenylisothiocyanate and methyl iodide in the presence of KOH giving the N, S-ketene acetal derivatives 7a and 7b.
Structure 7a was elucidated by its IR spectrum that showed two NH peaks at 3347 and 3291 cm -1 as well as a C≡N band at 2179 cm -1 and also its 1 H NMR spectrum that exhibited a singlet at δ 2.28 ppm (SCH3) and two D2O exchangeable peaks at δ 9.97 and 11.55 corresponding to (NHCO) and (NH phenyl) respectively.Also, 13 C NMR of 7a confirm the appearance of signals for (S-CH3), β-ketene and α-ketene at δ 16.89, 74.35, 167.91in sequence.These keteneacetals 7a and 7b were cyclized upon reaction with hydrazine in ethanol resulting in 8a and 8b.The structure 8 is postulated through its spectral data and subsequent reaction.The 1 H NMR of 8a showed a singlet D2O exchangeable peak at δ 6.11 corresponding to (NH2) group (Scheme1).Then, these 5-aminopyrazoles were cyclized to the target compounds 9a and 9b through their reaction with 2,4-pentanedione in boiling acetic acid.With a symmetric diketone, only one compound was formed, the reaction proceeded that either NH or NH2 reacted with any carbonyl followed by condensation and removal of 2 moles of water giving 9a and 9b (Scheme 2).
The structure 9 was elucidated by elemental analysis and spectral data.Its IR revealed the disappearance of the cyano group and 1 H NMR spectrum of 9b showed singlets at δ2.65, 2.69 for two (CH3) while pyrimidine H-5 appeared at 6.80-7.00 as a multiplet with H-4 of NHphenyl (Scheme 2).Additionally, the 5-aminopyrazoles 8a,b were reacted with β-ketoesters such as ethyl acetoacetate or ethyl 3-oxo-3-phenylpropanoate resulting in pyrazolopyrimidine compounds 10a-d (scheme3).The structure 10a-d was confirmed by elemental analysis, spectral data and MO calculation (Table1).
Theoretical MO calculations of heat of formation and bend energy (energy required to bend all bonds in a molecule that could be a measure of its stability) was done by using Chemdraw ultra and MM2 property revealed that 10a-d is more stable in form A than B. The MO caluculation of (10a,A) showed a heat of formation = -339.40086Kcal/mol and bend energy = 19.8561Kcal/mol, while in form B a heat of formation and bend energy = -369.39Kcal/mol and 20.508 Kcal/mol were found respectively.The previous result indicates that form A is more favorable than B. Practically, for 10a and due to lack of formation of a single crystal analysis, the structure of 10a was illustrated using HSQC, HMBC and NOESY techniques (Fig. 2).Upon studying 3D model of 10a in either form A or B, form A showed a correlation between the protons of methyl group and both of pyrimidine H, and C-4H of NH-phenyl in a distance within the range that appeared in the NOESY experiment (Fig. 2), while form B does not show these correlations.NOESY scan confirmed that 10a is in form A and not B.

Anticancer screening:
Cytotoxic study was operated using MTT(3,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide assay method. 21All tested compounds showed moderate to high cytotoxic activity against MCF-7, BT474 and A549 cell lines.The obtained data are listed Table 2. Most synthesized compounds (7a, 8a, 8b, 9a, 9b, 10a and 10c) showed moderate cytotoxic activity when compared with the reference.7bis the least potent one that exhibited a cytotoxic activity with IC509.02,9.87, 8.42 µM against MCF-7, BT474 and A549 cell lines.Regarding compounds with an oxazole moiety only (6a and 8a) they showed nearly the same moderate activity against all cell lines and they are more potent than (6b and 8b) with a benzothiazole moiety.The sight was directed to the compound of benzoythiazole or oxazole /pyrazolopyrimidine hybrids (9a, 9b and 10a-d).Their potency was ordered 10d >10b > 9b > 9a >10a>10c.The most potent is 10d with IC501.98,2.2, 2.61µM and its result is close to the reference doxorubicin with IC501.72,1.81, 1.21 µM against MCF-7, BT474 and A549 cell lines.10d is a pyrazolopyrimidine-benzothiazole hybrid and this reactivity could be related to, 1-high potency of benzothiazole moiety, 2-presence of NH tatuomeric OH that increase incidence of hydrogen bond (HB) with proposed receptor active site and 3-high lipophilicity due to phenyl moiety when compared to 10b which is also pyrazolopyrimidine-benzothiazole hybrid with a CH3 moiety.In addition 10b also showed higher potency activity with IC50= 2.01, 2.36, 2.47 µM against MCF-7, BT474 and A549 cell lines respectively.Several benzothiazole and oxazole compounds (6a, 6b, 7a,7b, 8a and 8b) were prepared and also pyrazolopyrimidine with a benzoxazole (9a, 10a and 10c) those showed considerable cytotoxic activity while their corresponding pyrazolpyrimidines with a benzothiazole (9b, 10b and 10d) showed the highest activity.Such pyrazolopyrimidine-benzothiazole combination is the successful one that resulted in 10b and 10d as the most active compounds.Structure elucidation of 10a with 2D and NOE excluded their structural form.

Experimental Section
General.Melting points were determined on an Electrothermal digital melting point apparatus and are uncorrected.IR spectra were recorded on an R 435 spectrophotometer (Middlton, Madison West, WI, USA) and values were reported in cm−1. 1 H-NMR and 13 C-NMR were carried out on Bruker Advance III 400 MHz spectrophotometer (Bruker BioSpin AG, Fällanden, Switzerland) for 1 H and 100 MHz for 13 C with BBFO Smart Probe and Bruker 400 AEON Nitrogen-Free Magnet, using TMS as an internal standard and chemical shifts were recorded in ppm on δ scale, Faculty of Pharmacy, Beni Suef University, Egypt.The electron impact (EI) mass spectra were recorded on a Hewlett Packard 5988 spectrometer (Palo Alto, CA, USA), Microanalyses for C, H and N were carried out on Perkin-Elmer 2400 analyzer (Perkin-Elmer, Norwalk, CT, USA) at the Micro analytical unit of Cairo University, Egypt, and all compounds were within ±0.4% of the theoretical values.Thin-layer chromatography (TLC) was performed on Merck (Darmstadt, Germany ) TLC aluminium sheets silica gel 60 F254 with detection by UV quenching at 254 nm to follow the course of reactions and to check the purity of products.All reagents and solvents were purified and dried by standard techniques.

Table 1 :
Molecular orbital (M.O) calculation of heat of formation (HF) and bend energy (BE) for expected 10a-d (structure A or structure B)