Multiple biological active 4-aminopyrazoles containing trifluoromethyl and their 4-nitroso-precursors: Synthesis and evaluation.

4-Nitroso-3-trifluoromethyl-5-alkyl[(het)aryl]pyrazoles were synthesized via one-pot nitrosation of 1,3-diketones or their lithium salts followed by treatment of hydrazines. Reduction of nitroso-derivatives made it possible to obtain 4-amino-3-trifluoromethylpyrazoles chlorides. According to computer-aided calculations, all synthesized compounds are expected to have acceptable ADME profile for drug design. Tuberculostatic, antibacterial, antimycotic, antioxidant and cytotoxic activities of the compounds were evaluated in vitro, while their analgesic and anti-inflammatory action was tested in vivo along with acute toxicity studies. N-Unsubstituted 4-nitrosopyrazoles were the most effective tuberculostatics (MIC to 0.36 µg/ml) and antibacterial agents against Streptococcus pyogenes (MIC to 7.8 µg/ml), Staphylococcus aureus,S. aureus MRSA and Neisseria gonorrhoeae (MIC to 15.6 µg/ml). 4-Nitroso-1-methyl-5-phenylpyrazole had the pronounced antimycotic action against a wide range of fungi (Trichophytonrubrum, T. tonsurans, T. violaceum, T. interdigitale, Epidermophytonfloccosum, Microsporumcanis with MIC 0.38-12.5 µg/ml). N-Unsubstituted 4-aminopyrazoles shown high radical-scavenging activity in ABTS test, ORAC/AAPH and oxidative erythrocyte hemolysis assays. 1-Methyl-5-phenyl-3-trifluoromethylpyrazol-4-aminium chloride revealed potential anticancer activity against HeLa cells (SI > 1351). The pronounced analgesic activity was found for 4-nitroso- and 4-aminopyrazoles having phenyl fragment at the position 5 in "hot plate" test. The most of the obtained pyrazoles had a moderate acute toxicity.

Estimation of nizatidine gastric nitrosatability and product toxicity via an integrated approach combining HILIC, in silico toxicology, and molecular docking.

The liability of the H2-receptor antagonist nizatidine (NZ) to nitrosation in simulated gastric juice (SGJ) and under WHO-suggested conditions was investigated for the first time. For monitoring the nitrosatability of NZ, a hydrophilic interaction liquid chromatography (HILIC) method was optimized and validated according to FDA guidance. A Cosmosil HILIC® column and a mobile phase composed of acetonitrile: 0.04 M acetate buffer pH 6.0 (92:8, v/v) were used for the separation of NZ and its N-nitroso derivative (NZ-NO) within 6 min with LODs of 0.02 and 0.1 µg/mL, respectively. NZ was found highly susceptible to nitrosation in SGJ reaching 100% nitrosation in 10 min, while only 18% nitrosation was observed after 160 min under the WHO-suggested conditions. The chemical structure of NZ-NO was clarified by ESI+/MS. In silico toxicology study confirmed the mutagenicity and toxicity of NZ-NO. Experiments evidenced that ascorbic acid strongly suppresses the nitrosation of NZ suggesting their co-administration for protection from potential risks. In addition, the impacts of the HILIC method on safety, health, and environment were favorably evaluated by three green analytical chemistry metrics and it was proved that, unlike the popular impression, HILIC methods could be green to the environment.

Synthesis and Biological Evaluation of N(2) -Substituted 2,4-Diamino-6-cyclohexylmethoxy-5-nitrosopyrimidines and Related 5-Cyano-NNO-azoxy Derivatives as Cyclin-Dependent Kinase†2 (CDK2) Inhibitors.

The potent and selective cyclin-dependent kinase 2 (CDK2) inhibitor NU6027 (6-cyclohexylmethoxy-5-nitroso-2,4-diaminopyrimidine) was used as the lead for the synthesis of a series of analogues in order to provide further insight into the structure-activity relationships for 2,4-diaminopyrimidine CDK2 inhibitors. Aliphatic amino substituents were introduced at position 2. The use of linear or less sterically hindered amines gave rise to compounds endowed with slightly better activity than the lead; on the other hand, the compounds were less active if a bulkier amino substituent was used. Substitution of the 5-nitroso group with a 5-cyano-NNO-azoxy moiety afforded a new class of inhibitors, the activity of which against CDK2 was found to be similar to that of the nitroso series. The most active nitroso compound was 8 b ((2S)-2-[(4-amino-6-cyclohexylmethoxy-5-nitrosopyrimidin-2-yl)amino]propan-1-ol; IC50 =0.16 µm), while in the 5-cyano-NNO-azoxy series the most active compound was 9 b (4-amino-5-[(Z)-cyano-NNO-azoxy]-2-{[(2S)-1-hydroxypropan-2-yl]amino}-6-cyclohexylmethoxypyrimidine; IC50 =0.30 µm). Taken together, these new analogues of NU6027 enhance our understanding of the structure-activity relationships for 2,4-diaminopyrimidine CDK2 inhibitors.

Carbonic anhydrases are producers of S-nitrosothiols from inorganic nitrite and modulators of soluble guanylyl cyclase in human platelets.

Nitric oxide (NO), S-nitrosoglutathione (GSNO) and S-nitrosocysteine are highly potent signaling molecules, acting both by cGMP-dependent and cGMP-independent mechanisms. The NO metabolite nitrite (NO2 (-)) is a major NO reservoir. Hemoglobin, xanthine oxidoreductase and carbonic anhydrase (CA) have been reported to reduce/convert nitrite to NO. We evaluated the role and the physiological importance of CA for an extra-platelet CA/nitrite/NO/cGMP pathway in human platelets. Authentic NO was analyzed by an NO-sensitive electrode. GSNO and GS(15)NO were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). cGMP was determined by LC-MS/MS or RIA. In reduced glutathione (GSH) containing aqueous buffer (pH 7.4), human and bovine erythrocytic CAII-mediated formation of GSNO from nitrite and GS(15)NO from (15)N-nitrite. In the presence of L-cysteine and GSH, this reaction was accompanied by NO release. Incubation of nitrite with bovine erythrocytic CAII and recombinant soluble guanylyl cyclase resulted in cGMP formation. Upon incubation of nitrite with bovine erythrocytic CAII and washed human platelets, cGMP and P-VASP(S239) were formed in the platelets. This study provides the first evidence that extra-platelet nitrite and erythrocytic CAII may modulate platelet function in a cGMP-dependent manner. The new nitrite-dependent CA activity may be a general principle and explain the cardioprotective effects of inorganic nitrite in the vasculature. We propose that nitrous acid (ONOH) is the primary CA-catalyzed reaction product of nitrite.

Structural and theoretical studies on rhodium and iridium complexes with 5-nitrosopyrimidines. Effects on the proteolytic regulatory enzymes of the renin-angiotensin system in human tumoral brain cells.

The reactions of [RhCl(CO)(PPh3)2], [RhCl(CO)2]2 and [IrCl(CO)(PPh3)2] with different 5-nitrosopyrimidines afforded sixteen complexes which have been structurally characterized by elemental analysis, IR and NMR ((1)H and (13)C) spectral methods and luminescence spectroscopy. The crystal and molecular structures of [Rh(III)Cl(VIOH-1)2(PPh3)], [Rh(III)Cl(DVIOH-1)2(PPh3)] and [Rh(II)(DVIOH-1)2(PPh3)2] have been established from single crystal x-ray structure analyses. The three complexes are six-coordinated with both violurato ligands into an equatorial N5,O4-bidentate fashion, but with different mutually arrangements. Theoretical studies were driven on the molecular structure of [Rh(III)Cl(VIOH-1)2(PPh3)] to assess the nature of the metal-ligand interaction as well as the foundations of the cis-trans (3L-2L) isomerism. An assortment of density functional (SOGGA11-X, B1LYP, B3LYP, B3LYP-D3 and wB97XD) has been used, all of them leading to a similar description of the target system. Thus, a topological analysis of the electronic density within AIM scheme and the study of the Mulliken charges yield a metal-ligand link of ionic character. Likewise, it has been proved that the cis-trans isomerism is mainly founded on that metal-ligand interaction with the relativistic effects playing a significant role. Although most of the compounds showed low direct toxicity against the human cell lines NB69 (neuroblastoma) and U373-MG (astroglioma), they differently modify in several ways the renin-angiotensin system (RAS)-regulating proteolytic regulatory enzymes aminopeptidase A (APA), aminopeptidase N (APN) and insulin-regulated aminopeptidase (IRAP). Therefore, these complexes could exert antitumor activity against both brain tumor types, acting through the paracrine regulating system mediated by tissue RAS rather than exerting a direct cytotoxic effect on tumor cells.

Antiproliferative effects of palladium(II) complexes of 5-nitrosopyrimidines and interactions with the proteolytic regulatory enzymes of the renin-angiotensin system in tumoral brain cells.

Seventeen new palladium(II) complexes of general formulaes PdCl2L, PdCl(LH-1)(solvent) and PdCl2(PPh3)2L containing pyrimidine ligands derived from 6-amino-5-nitrosouracil and violuric acid have been prepared and characterized by elemental analysis, IR and NMR ((1)H and (13)C) methods and, two of them, PdCl(DANUH-1)(CH3CN)]·½H2O and [PdCl(2MeOANUH-1)(CH3CN)] by X-ray single-crystal diffraction (DANU: 6-amino-1,3-dimethyl-5-nitrosouracil; 2MeOANU: 6-amino-2-methoxy-5-nitroso-3H-pyrimidin-4-one). The coordination environment around palladium is nearly square planar in the two compounds with different supramolecular arrangements. Crystallographic and spectral data are consistent with a bidentate coordination mode through N5 and O4 atoms when the ligands act in neutral form and N5 and N6 atoms in the monodeprotonated ones. The cytotoxicity of the complexes against human neuroblastoma (NB69) and human glioma (U373-MG) cell lines has been tested showing a considerable antiproliferative activity. Also, the study of the effects of palladium(II) complexes on the renin-angiotensin system (RAS) regulating proteolytic regulatory enzymes aminopeptidase A (APA), aminopeptidase N (APN) and insulin-regulated aminopeptidase (IRAP) shows a strong dependence on the compound tested and the tumoral cell type, also affecting different catalytic routes; the compounds affect in a different way the activities of enzymes of the RAS system, changing their functional roles as initiators of cell proliferation in tumors as autocrine/paracrine mediators.

Dioxygen mediated conversion of {Fe(NO)2}9 dinitrosyl iron complexes to Roussin's red esters.

The reactivity of a series of {Fe(NO)2}(9) dinitrosyl iron complexes bearing thiolate ligands with molecular oxygen is reported. These reactions result in the formation of the corresponding Roussin's red esters along with thiolate oxidation. This reactivity is contrasted with that previously reported for {Fe(NO)2}(10) complexes.

Cellular uptake mechanisms and responses to NO transferred from mono- and poly-S-nitrosated human serum albumin.

Endogenous S-nitrosated human serum albumin (E-Mono-SNO-HSA) is a large molecular weight nitric oxide (NO) carrier in human plasma, which has shown many beneficial effects in different animal models. To construct more efficient SNO-HSA preparations, SNO-HSA with many conjugated SNO groups has been prepared using chemical modification (CM-Poly-SNO-HSA). We have compared the properties of such a preparation to those of E-Mono-SNO-HSA. Cellular uptake of NO from E-Mono-SNO-HSA partly takes place via low molecular weight thiol, and it results in cytoprotective effects by induction of heme oxygenase-1. By contrast, transfer of NO from CM-Poly-SNO-HSA into the cells is faster and more pronounced. The influx mainly takes place by cell-surface protein disulfide isomerase. The considerable NO inflow results in apoptotic cell death by ROS induction and caspase-3 activation. Thus, increasing the number of SNO groups on HSA does not simply intensify the cellular responses to the product but can also result in very different effects.

Dye-tethered ruthenium nitrosyls containing planar dicarboxamide tetradentate N4 ligands: effects of in-plane ligand twist on NO photolability.

To examine the steric effects of the in-plane ligands in dye-sensitized {RuNO}(6) nitrosyls on their NO photolability, two new ligands, namely, 1,2-Bis(pyridine-2-carboxamido)-4,5-dimethoxybenzene (H(2)(OMe)(2)bpb) and 1,2-Bis(Isoquinoline-1-carboxamido)-4,5-dimethoxybenzene (H(2)(OMe)(2)IQ1, H's are dissociable carboxamide protons) have been designed and synthesized. The syntheses and spectroscopic properties of {RuNO}(6) nitrosyls derived from these two ligands, namely, [((OMe)(2)bpb)Ru(NO)(Cl)] (4-Cl), [((OMe)(2)IQ1)Ru(NO)(Cl)] (5-Cl), [((OMe)(2)bpb)Ru(NO)(Resf)] (4-Resf), and [((OMe)(2)IQ1)Ru(NO)(Resf)] (5-Resf), are reported. The structures of 5-Cl, 4-Resf, and 5-Resf have been determined by X-ray crystallography. Removal of the in-plane ligand twist in the quinoline-based R(2)bQb(2-) ligand frame (because of steric interactions between the extended quinoline ring systems) in both R(2)bpb(2-) and R(2)IQ1(2-) (pyridine and 1-isoquinoline rings, respectively, instead of quinoline rings in the equatorial plane) results in enhanced solution stability, as well as higher quantum yield values for NO photorelease upon exposure to 500 nm light. Both dye-tethered {RuNO}(6) nitrosyls 4-Resf and 5-Resf exhibit greater sensitivity to visible light compared to the chloro-bound species 4-Cl and 5-Cl. In addition, the dye-tethered nitrosyls are fluorescent and hence can be used as trackable NO donors in cellular studies.

The water-soluble Roussin's red ester acting as a potential photochemical NO-delivery agent: photolysis reactions, DNA cleavage and anticancer activity.

The water-soluble Roussin's red ester [(NO)(2)Fe(mu-SCH(2)CH(2)P(O)(CH(2)OH)(2))(2)Fe(NO)(2)] (1), a potential photochemical prodrug of an NO precursor, was synthesized from the reaction of HSCH(2)CH(2)P(O)(CH(2)OH)(2) (F) and [Fe(CO)(2)(NO)(2)]. The IR v(NO) stretching frequencies of complex 1 appear at 1759 (s), 1784 (s) and 1816 (w) cm(-1) in buffer (pH = 7.4). NO was released with a stoichiometry ratio Delta[NO]/Delta[1] = 3.6 +/- 0.2 when complex 1 was exposed to UV in deaerated aqueous phosphate buffer solution. Here light acts as an On/Off switch for NO release. Incubation of pBR322 supercoiled DNA with complex 1, followed by irradiation, produced DNA strand breakage. In contrast to the addition of carboxy-PTIO (NO radical scavenger), DNA strand breakage was not inhibited when the scavengers of hydroxyl radical and singlet oxygen were added. Complex 1 irradiated under a N(2) atmosphere exhibited the same cleavage efficiency as complex 1 irradiated under air. The results show that DNA strand cleavage efficiency depends on the concentration of complex 1, the pH value of the buffer, and the duration of the photolysis of complex 1. The conversion rate from supercoiled (SC form) to nicked circular (NC form) of complex 1 was 2.96 x 10(-2) s(-1). The results of a T4 ligase enzymatic assay reveals the nonhydrolytic DNA breakage mechanism. The NO-release ability of complexes 1, 2, and 3 follows the order 1 > 2 > 3. Upon UV-irradiation, complex 1 exhibits cytotoxicity against B16-F10 mouse melanoma cells.

N-nitrosomelatonin: synthesis, chemical properties, potential prodrug.

Melatonin is easily nitrosated via various mechanisms at the nitrogen atom of the indole ring to give N-nitrosomelatonin (NOMela). This mini-review provides a comprehensive view of this N-nitroso compound. With an improved procedure NOMela can now economically synthesized with low laboratory expenditure. The major chemical property of NOMela, i.e. the (formally) transfer of the NO+ function to its target nucleophile, is explained in detail and a variety of detection methods using this reaction are suggested. As the suspected carcinogenical potential of NOMela is clearly overruled it seems attractive to apply this nitroso compound for endogenous generation of S-nitrosothiols that act as nitric oxide donors in vivo.

A diazonium ion cascade from the nitrosation of tolazoline, an imidazoline-containing drug.

Tolazoline (1-benzylimidazoline), a representative imidazoline-containing drug, reacts readily with nitrite in acetic acid to produce a complex product mixture. Fourteen compounds have been identified as products of this transformation when an 8-fold excess of HNO2 is used. The products, which include N-nitrosoamides, esters, alcohols, and phenylacetic acid, are rationalized as arising from a cascade of reactive diazonium ions. N-Nitrosotolazoline can be isolated from the nitrosation reaction in good yield when the mixture is extracted with CH2Cl2 as the transformation progresses. It nitrosates much more rapidly (50x) than tolazoline to give, among other products, the oxime [1-( N-nitroso-2-imidazolinyl)benzylidene]hydroxylamine, which can also be produced in good yield from the reaction of tolazoline with isopropyl nitrite. At low substrate and nitrite concentrations, the main reaction products are N-nitrosotolazoline, its decomposition product N-2-hydroxyethylphenylacetamide, the above-mentioned oxime, phenyl acetic acid, and 2-hydroxyethyl phenylacetate. The tolazoline nitrosation rate in three buffer systems has been determined at pH 3.4 and 37 degrees C ( kobs = 6.25 x 10 (-5) s (-1) in 0.5 M acetate buffer with a 10 * [NO2(-)] = 250 mM). Because N-nitrosotolazoline exhibits the chemical properties of a direct-acting mutagen and carcinogen, we have used the rate data to estimate its level of formation at nitrite concentrations <3 mM. Cursory examination of the nitrosation chemistry of oxymetazoline, a related drug, is primarily focused at its electron-rich aromatic ring.

Design and evaluation of S-nitrosylated human serum albumin as a novel anticancer drug.

In recent studies, the cytotoxic activity of NO has been investigated for its potential use in anticancer therapies. Nitrosated human serum albumin (NO-HSA) may act as a reservoir of NO in vivo. However, there are no published reports regarding the effects of NO-HSA on cancer. Therefore, the present study investigated the antitumor activity of NO-HSA. NO-HSA was prepared by incubating HSA, which had been sulfhydrylated using iminothiolane, with isopentyl nitrite (6.64 mol NO/mol HSA). Antitumor activity was examined in vitro using murine colon 26 carcinoma (C26) cells and in vivo using C26 tumor-bearing mice. Exposure to NO-HSA increased the production of reactive oxygen species in C26 cells. Flow cytometric analysis using rhodamine 123 showed that NO-HSA caused mitochondrial depolarization. Activation of caspase-3 and DNA fragmentation were observed in C26 cells after incubation with 100 muM NO-HSA for 24 h, and NO-HSA inhibited the growth of C26 cells in a concentration-dependent manner. The growth of C26 tumors in mice was significantly inhibited by administration of NO-HSA compared with saline and HSA treatment. Immunohistochemical analysis of tumor tissues demonstrated an increase in terminal deoxynucleotidyl transferase dUTP nickend labeling-positive cells in NO-HSA-treated mice, suggesting that inhibition of tumor growth by NO-HSA was mediated through induction of apoptosis. Biochemical parameters (such as serum creatinine, blood urea nitrogen, aspartate aminotransferase, and alanine aminotransferase) showed no significant differences among the three treatment groups, indicating that NO-HSA did not cause hepatic or renal damage. These results suggest that NO-HSA has the potential for chemopreventive and/or chemotherapeutic activity with few side effects.

Synthesis and solid-state molecular structures of nitrosoalkane complexes of iron porphyrins containing methanol, pyridine, and 1-methylimidazole ligands.

Nitrosoalkanes belong to the family of C-nitroso compounds and are known to bind to the iron center in heme proteins. We have prepared and characterized a series of new nitrosoalkane heme model complexes of the form (por)Fe(RNO)(L) (por=porphyrinato dianion; R=isopropyl; L=MeOH, pyridine, 1-methylimidazole) by infrared and 1H NMR spectroscopy and X-ray crystallography. Within the set of octaethylporphyrinato (OEP) compounds, the infrared stretching frequencies of the NO groups decrease in the order (OEP)Fe(iPrNO)(MeOH).MeOH (1433 cm-1) > (OEP)Fe(iPrNO)(py) (1429 cm-1) > (OEP)Fe(iPrNO)(1-MeIm) (1423 cm-1), reflecting the increased backdonation of electron density in the 1-methylimidazole derivative. The molecular structures of the compounds as determined by crystallography reveal N-binding of the nitrosoalkane ligands to the formally ferrous metal centers.

Combination of paclitaxel and nitric oxide as a novel treatment for the reduction of restenosis.

The combination of a nitric oxide (NO) donor and a paclitaxel-NO donor conjugate coated on a vascular stent was tested in a rabbit iliac artery model of stenosis as a potential therapy for restenosis. Paclitaxel was conjugated with a NO donor at the 7-position to give compound 7. An adamantane-based NO donor 14 was synthesized and combined with 7 to provide a burst of NO in the first few critical hours following injury to the vessel wall. Both 7 and 14 demonstrated antiproliferative activity (IC(50) = 20 nM and 15 microM, respectively) and antiplatelet activity (IC(50) = 10 and 1 microM, respectively). Stents were coated with a layer of a polymer containing test compounds. The total amount of NO eluted from the stents after a 6 h implantation in the rabbit iliac artery was 35%, 95%, and 69% of the original content for the stents coated with 7, 14, and the combination of 7 and 14, respectively. The antistenotic activity of 7 and 14 was determined in a 28-day rabbit model with two control groups (uncoated stents and polymer-coated stents) and two study groups (paclitaxel-coated stents and stents coated with the combination of 7 and 14). Polymer-coated stents caused inflammation and increased stenosis by 39% when compared to the uncoated stents. The stents coated with 7 plus 14 were as good as the uncoated stents, 41% better than the polymer-coated stents and 34% better than the paclitaxel-coated stents. These data indicate a beneficial effect of adding NO to an antiproliferative agent (paclitaxel) and suggest a potential therapeutic combination for the treatment of stenotic vessel disease.

Inclusion complexes of 2-chloroethylnitrososulfamides (CENS) with beta-cyclodextrin.

Host-guest association between four CENS (derivated from piperidine, dibenzylamine, dicyclohexylamine and methyl prolinate) and beta-cyclodextrin was carried out in solution and solid state. Characterisation, stoichiometry, solubility, dissociation constants and stability of such complexes were studied, showing that the inclusion with beta-CD appears as a promising mode of formulation of 2-chloroethylnitrososulfamides.

Five- to six-coordination in (nitrosyl)iron(II) porphyrinates: effects of binding the sixth ligand.

We report structural and spectroscopic data for a series of six-coordinate (nitrosyl)iron(II) porphyrinates. The structures of three tetraphenylporphyrin complexes [Fe(TPP)(NO)(L)], where L = 4-(dimethylamino)pyridine, 1-methylimidazole, 4-methylpiperidine, are reported here to a high degree of precision and allow observation of several previously unobserved structural features. The tight range of bonding parameters for the [FeNO] moiety for these three complexes suggests a canonical representation for six-coordinate systems (Fe-N(p) = 2.007 A, Fe-N(NO) = 1.753 A, angle FeNO = 138.5 degrees ). Comparison of these data with those obtained previously for five-coordinate systems allows the precise determination of the structural effects of binding a sixth ligand. These include lengthening of the Fe-N(NO) bond and a decrease in the Fe-N-O angle. Several other aspects of the geometry of these systems are also discussed, including the first examples of off-axis tilting of a nitrosyl ligand in a six-coordinate [FeNO](7) heme system. We also report the first examples of Mössbauer studies for these complexes. Measurements have been made in several applied magnetic fields as well as in zero field. The spectra differ from those of their five-coordinate analogues. To obtain reasonable fits to applied magnetic field data, rotation of the electrical field gradient is required, consistent with differing g-tensor orientations in the five- vs six-coordinate species.

4-Alkoxy-2,6-diaminopyrimidine derivatives: inhibitors of cyclin dependent kinases 1 and 2.

The cyclin dependent kinase (cdk) inhibitor NU6027, 4-cyclohexylmethoxy-5-nitroso-pyrimidine-2,6-diamine (IC(50) vs cdk1/cyclinB1=2.9+/-0.1 microM and IC(50) vs cdk2/cyclinA3=2.2+/-0.6 microM), was used as the basis for the design of a series of 4-alkoxy-2,6-diamino-5-nitrosopyrimidine derivatives. The synthesis and evaluation of 21 compounds as potential inhibitors of cyclin-dependent kinases 1 and 2 is described and the structure-activity relationships relating to NU6027 have been probed. Simple alkoxy- or cycloalkoxy-groups at the O(4)-position were tolerated, with the 4-(2-methylbutoxy)-derivative (IC(50) vs cdk1/cyclinB1=12+/-2 microM and cdk2/cyclinA3=13+/-4 microM) retaining significant activity. Substitutions at the N(6) position were not tolerated. Replacement of the 5-nitroso substituent with ketone, oxime and semicarbazone groups essentially abolished activity. However, the derivative bearing an isosteric 5-formyl group, 2,6-diamino-4-cyclohexylmethoxy-pyrimidine-5-carbaldehyde, showed modest activity (IC(50) vs cdk1/cyclinB1=35+/-3 microM and cdk2/cyclinA3=43+/-3 microM). The X-ray crystal structure of the 5-formyl compound bound to cdk2 has been determined to 2.3A resolution. The intramolecular H-bond deduced from the structure with NU6027 bound to cdk2 is not evident in the structure with the corresponding formyl compound. Thus the parent compound, 4-cyclohexylmethoxy-5-nitrosopyrimidine-2,6-diamine (NU6027), remains the optimal basis for future structure-activity studies for cyclin-dependent kinase inhibitors in this series.

4-Amino-6-benzyloxy-2-(methylsulfanyl)-5-nitrosopyrimidine: hydrogen-bonded dimers linked into pi-stacked chains.

The title compound, C(12)H(12)N(4)O(2)S, crystallizes with Z' = 2 in space group P2(1)/c. The intramolecular dimensions are consistent with a highly polarized electronic structure. Each of the independent molecules forms a centrosymmetric dimer linked by paired N-H.N hydrogen bonds, and these dimers are linked into a single type of chain by aromatic pi-pi-stacking interactions.