Synthesis and antimitotic properties of ortho -substituted polymethoxydiarylazolopyrimidines

Ortho -substituted polymethoxydiarylazolopyrimidines were synthesized using polymethoxysubstituted benzaldehydes and acetophenones as starting material. X-ray crystallography data clearly confirmed that the subsequent cyclization of 3-amino-1,2,4-triazole with ketoaldehydes yielded polymethoxyphenylsubstituted 6,7-diaryl-[1,2,4]triazolo[1,5-a ]pyrimidines as single isomers. All compounds were evaluated in vivo using phenotypic sea urchin embryo assay. 6-(4-Methoxyphenyl)-7-(3,4,5-trimethoxyphenyl)pyrazolo[


Introduction
The pyrimidine core, a building unit of DNA, RNA and nucleosides, can be found in a number of natural products representing pharmacologically attractive chemical scaffold.Various pyrimidine derivatives were reported to exhibit diverse biological activities including antimicrobial, 1,2 antitubercular, 3 antifungal, 4 antiviral, 5 and antitumor effects. 6,7Notably, polymethoxysubstituted diarylpyrimidines displayed dual antimicrobial-anticancer properties.As an example, a pyrimidin-2-one (Figure 1, I) demonstrated antifungal activity against Candida albicans comparable with that of clotrimazole as well as inhibited growth of a panel of 60 human cancer cell lines with mean GI50, TGI and LC50 values of 3.39, 17.4, and 61.7 microMol, respectively.Diarylpyrimidines can be considered as structural analogues of a potent natural antimitotic combretastatin A-4 (CA4, Figure 1, II) isolated from the bark of African willow tree Combretum caffrum. 9The cis-configuration of CA4 is essential for its antiproliferative antitubulin effect.Spontaneous isomerization to the trans-double bond observed both in vitro and in vivo causes a dramatic decrease of biological activity. 10To stabilize the active cis-conformation, several heteroaromatic rings, such as pyrazole, 11 imidazole, 11,12 thiazole, 11 isoxazole, 13,14 1,2,3-thiadiazole, 15 isomeric triazoles, 11,16,17 and tetrazole 11,[18][19][20] have been introduced as a nonisomerizable and metabolically stable isosteric equivalent of cis-double bond (Figure 1, III), resulting in highly active microtubule destabilizing antimitotic agents.Replacement of the double bond by triazolopyrimidine yielded diaryl-o-substituted-triazolopyrimidines (Figure 1, IV) with pronounced cytotoxicity against human cancer cells both in vitro and in mouse xenograft model. 21However, in contrast to CA4 derivatives III, these compounds promoted purified tubulin polymerization and stimulated aggregation of mitotic spindle microtubules in cells. 21or the design of potential anticancer molecules we synthesized a series of ortho-substituted polymethoxydiarylpyrimidines (Scheme 1, 7,8,9aa).4-Methoxy, 3,4,5-trimethoxy, and 3-hydroxy-4-methoxy substituted aromatic aldehydes and ketones were used as starting material to prepare azolopyrimidines structurally similar to CA4 and its potent heterocyclic derivatives.The resulting compounds were evaluated for antimitotic activity in a sea urchin embryo model.Selected molecules were also screened against a panel of human cancer cell lines (NCI60 anticancer drug screen, http://dtp.cancer.gov).
Type A: Connection of the aldehyde group with the amino group and of the ketone moiety with N1 atom. 25][28] Type C: Binding of the aldehyde group with the amino group and of the ketone moiety with N4 atom.Up to date, there are no literature data on the formation of a pure type C isomer.Type D: Connection of the aldehyde group with N4 atom and of the ketone moiety with the amino group. 28he formation of mixtures of two isomers A+B 26,27 or A+C 29 was published as well.Only two examples of diarylketoaldehydes cyclization with 3-amino-1,2,4-triazoles that proceeded according to type A 21,27 or D 30 have been reported.
X-ray structure determination.Literature data [25][26][27][28][29][30] together with our results suggest that the structure of key pyrimidine products 9 could not be unambiguously confirmed by NMR analysis.In the present study X-ray crystallography was carried out in order to determine the precise structure of 6-(4-methoxyphenyl)-7-(3,4,5trimethoxyphenyl) [1,2,4]triazolo [1,5-a]pyrimidine (9aa).The analysis confirmed type A cyclization with the formation of compound 9aa as isomer A (Figure 2).The yellow prismatic crystal of 9aa (C21H20N4O4, M = 392.41)was triclinic, space group, P-1, at T = 120 K: a ) and corrected for absorption. 31The structure was solved by direct methods and refined by a full-matrix least squares technique on F 2 with anisotropic displacement parameters for non-hydrogen atoms.One of the eight methoxy groups was disordered over two sites with the occupancies of 0.8:0.2.The hydrogen atoms were placed in calculated positions and refined within the riding model with fixed isotropic displacement parameters (Uiso(H) = 1.5Ueq(C) for the CH3-group and Uiso(H) = 1.2Ueq(C) for the other groups).The final divergence factors were R1 = 0.049 for 7812 independent reflections with I > 2(I) and wR2 = 0.132 for all independent reflections, S = 1.010.All calculations were carried out using the SHELXTL program. 32Full crystallographic data for compound 9aa (Tables S1-S6, Supplementary Material) have been deposited with the Cambridge Crystallographic Data Center, CCDC 1525158.Copies of this information may be obtained free of charge from the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK. 33 Biological evaluation.The targeted diarylazolopyrimidines 7-9 and by-product 6 were evaluated for antimitotic activity in a phenotypic sea urchin embryo assay. 34The assay provides rapid information on antiproliferative, embryotoxic, and microtubule destabilizing activities of tested molecules.The results are presented in Table 1 with CA4 as a positive control.For comparison, EC and GI50 values for intermediate chalcones 3aa, 3ab, 3d, and 3e were included.Compounds 3d and 3e were synthesized as described in the Experimental Section.As shown from Table 1, intermediate chalcones 3aa and 3ab exhibited antimitotic activity in the sea urchin embryo assay.Embryo spinning after the exposure to 3ab, a full analog of CA4, and tuberculate eggs characteristic for 3aa suggested their microtubule destabilizing mode of action.Notably, the interchange of substituted aryl rings in chalcones 3aa and 3ab yielded significantly less active compounds 3d and 3e, respectively.Therefore we paid attention to the synthesis of azolopyrimidine isomers 8, 9aa, and 9ab from intermediate chalcones 3aa and 3ab.Cyclization of chalcones 3 into dyarylazolopyrimidines 8 and 9 resulted in decrease of antimitotic effect.Only pyrazolopyrimidine 8 exhibited antiproliferative and cytotoxic effects both in sea urchin embryos and human cancer cells.Compound 8 could be considered as microtubule targeting agent since it caused formation of tuberculate arrested eggs typical for microtubule destabilizers.In contrast to microtubule stabilizing diaryl-o-substituted-triazolopyrimidines (Figure 1, IV), 21 the antimitotic effect of pyrazolopyrimidine 8 was likely associated with microtubule disassembly.Pyrimidine derivative 7 as well as by-product 6 displayed weak antiproliferative activity.All four triazolopyrimidines 9 failed to affect cell division (cleavage) up to 4 microMol concentration.2][13][14][15][16][17][18][19][20] Similarly, CA4 analogues with bicyclic triazolopyrimidine linker, halogen substituted triazolopyrimidines (Figure 1, IV), displayed cytotoxicity against human cancer cell lines with IC50 in nanomolar concentration range. 21In the present study, polymethoxyhydroxy-substituted diaryltriazolopyrimidines 9 were found to be inactive up to 4 microMol concentration.The results indicated the importance of aryl rings substituents in diaryltriazolopyrimidine derivatives of CA4 for their antimitotic antitubulin effects.

Experimental Section
General.Melting points were measured on a Boetius melting point apparatus and were uncorrected.Reaction mixtures were stirred magnetically. 1H NMR spectra were recorded on a Bruker DRX-500 (500.13MHz) instrument.Chemical shifts were stated in parts per million (ppm) and referenced to the appropriate NMR solvent peak(s).Spin-spin coupling constants (J) were reported in hertz (Hz).Original 1 H NMR spectra for azolopyrimidines 7, 8, and 9 are presented in Supplementary Material.Low resolution mass spectra (m/z) were recorded on a Finnigan MAT/INCOS 50 mass spectrometer at 70 eV using direct probe injection.Elemental analysis was performed on the automated PerkinElmer 2400 CHN microanalyzer.Flash chromatography was carried out on silica gel (Acros 0.035-0.070mm, 60 Å).TLC was performed on Merck 60 F254 plates.
General procedure for the synthesis of chalcones 3. 37 NaOH (60 mmol) was added to a vigorously stirred solution containing arylacetophenones 1 (20 mmol) and benzaldehydes 2 (20 mmol) in EtOH (150 mL) at 5 C.The reaction mixture was stirred at room temperature for 24 h and the solvent was evaporated.The residue was diluted with water (130 mL), neutralized with 15% HCl, and extracted with CH2Cl2 (3  80 mL).Organic extracts were washed with brine (2  80 mL), dried with Na2SO4, and evaporated.The residue was crystallized from EtOAc-petroleum (1:1) and dried to afford chalcones 3.

Table 1 .
Effects of diarylazolopyrimidines on sea urchin embryos and human cancer cells 34The sea urchin embryo assay was conducted as described previously.34Fertilizedeggsandhatched blastulae were exposed to 2-fold decreasing concentrations of compounds.Duplicate measurements showed no differences in effective threshold concentration (EC) values.bI50: concentration required for 50% cell growth inhibition.c ECalues from Ref.35.d TE: tuberculate eggs typical of microtubule destabilizing agents.e Data from Ref. 36 for K562 human leukemia cell line.f ND: not determined.g Mean value for 60 human cancer cell lines.h Mean value for 60 human cancer cell lines, NSC 613729.