Synthesis, In Silico and In Vitro Cytostatic Activity of New Lipophilic Derivatives of Hydroxyurea.

BACKGROUND: Hydroxyurea (HU) is used to treat cancer. HU has a short half-life due to its small molecular weight and high polarity, therefore a high dosage of the drug should be used which introduces side effects and more rapid development of resistance. OBJECTIVE: The objective of the current study is to design new lipophilic analogues of hydroxyurea with higher stability and better cell penetration. The designed compounds were synthesized and then evaluated in terms of their cytostatic activities against two human cell lines. METHODS: The synthesis of designed ligands was achieved via two-step procedure. Detail of the synthesis and chemical characterization of the analogs are described. The cytotoxic activity of the designed ligands was evaluated in vitro against two different cancer cell lines at 24 and 48h using MTT test. RESULTS: Based on the IC50 values, all the designed and prepared compounds were more potent than hydroxyurea at 24 and 48h on both cell lines that the cytostatic activity at 48h was more than 24h. Drug-receptor interactions study indicated compound 7 as the most potent ligand, tightly bonded to surrounding amino acids in the active site of receptor via two strong hydrogen bonds and some hydrophobic interactions. CONCLUSION: Compound 7 with the suitable volume, log p and shape is the most active ligand against both cell lines. It is concluded or suggested that the size, shape and hydrophobic character of substituents strongly affect the pharmacodynamics and pharmacokinetics of these type of ligands.

A thalidomide-hydroxyurea hybrid increases HbF production in sickle cell mice and reduces the release of proinflammatory cytokines in cultured monocytes.

Fetal hemoglobin (HbF) induction by hydroxyurea (HU) therapy is associated with decreased morbidity and mortality in sickle cell anemia (SCA) patients, but not all patients respond to or tolerate HU. This provides a rationale for developing novel HbF inducers to treat SCA. Thalidomide analogs have the ability to induce HbF production while inhibiting the release of tumor necrosis factor-alpha. Molecular hybridization of HU and thalidomide was used to synthesize 3- (1,3-dioxoisoindolin-2-yl) benzyl nitrate (compound 4C). In this study, we show that compound 4C increases HbF production in a transgenic SCA mouse model and reduces the production of pro-inflammatory cytokines by SCA mouse monocytes cultured ex vivo. Therefore, compound 4C is a novel drug designed to treat SCA with a unique combination of HbF-inducing and anti-inflammatory properties.

Antiradical, chelating and antioxidant activities of hydroxamic acids and hydroxyureas.

Reactive oxygen species, along with reactive nitrogen species, may play an important role in the pathogenesis and progress of many diseases, including cancer, diabetes and sickle cell disease. It has been postulated that hydroxyurea, one of the main treatments in sickle cell disease, achieves its activity partly also through its antioxidant properties. A series of hydroxyurea derivatives of L- and D-amino acid amides and cycloalkyl-N-aryl-hydroxamic acids was synthesized and investigated for their radical scavenging activity, chelating properties and antioxidant activity. All the compounds showed exceptional antiradical activities. For example, free radical scavenging activities of investigated hydroxyureas were higher than the activity of standard antioxidant, butylated hydroxyanisole (BHA). Moreover, most of the investigated hydroxamic acids were stronger Fe²âº ion chelators than quercetin. In addition, the investigated compounds, especially hydroxamic acids, were proven to be excellent antioxidants. They were as effective as BHA in inhibiting ß-carotene-linoleic acid coupled oxidation. It is reasonable to assume that the antioxidant activity of the investigated compounds could contribute to their previously proven biological properties as cytostatic and antiviral agents.

Synthesis, antitumor evaluation and crystal structure of hydroxyurea derivatives.

A series of hydroxyurea derivatives have been synthesized and elucidated by means of FT-IR, (1)H-, (13)C-NMR and MS. The exact stereostructures of representative compounds have been determined by X-ray crystal structure analysis. In the crystals, inversion dimers linked by pairs of N-H...O hydrogen bonds occurred, and further N-H...O links led to chains of molecules. In vitro antitumor activities against Tca8113 human tongue cancer cells and L1210 murine leukemia cells were evaluated. A total of 8 of the 12 compounds had higher inhibitory activities than hydroxyurea against L1210 cells. Among them, the most promising compounds were 3e, 3d, 3a and 2d.

Neuroprotective effects of N-alkyl-1,2,4-oxadiazolidine-3,5-diones and their corresponding synthetic intermediates N-alkylhydroxylamines and N-1-alkyl-3-carbonyl-1-hydroxyureas against in vitro cerebral ischemia.

Herein we report the synthesis and neuroprotective effects of new N-alkyl-1,2,4-oxadiazolidine-3,5-diones and their corresponding synthetic intermediates, N-alkylhydroxylamines and N-1-alkyl-3-carbonyl-1-hydroxyureas, in an in vitro model of ischemia. We found five analogues that protect HT22 cells from death in the concentration range of 1-5 muM. Because members of the MAP kinase family are known to be key players in nerve cell survival and death, we characterized the role of these kinases in the neuroprotective mechanisms of the newly synthesized analogues. The results indicate that these compounds provide neuroprotection through distinct mechanisms of action.

Novel lipophilic hydroxyurea derivatives: synthesis, cytostatic and antiviral activity evaluations.

The novel hydroxyurea derivatives of l- and d-amino acid amides 5a-l were prepared by aminolysis of N-(1-benzotriazolecarbonyl)-amino acid amides 4a-f with O-benzylhydroxylamine and N-methylhydroxylamine and evaluated for their antiviral and cytostatic activity against malignant tumor cell lines and normal human fibroblasts. Compounds 5a, 5c, 5e and 5k showed the highest antiproliferative effects against all tumor cell lines tested. The strongest non-specific cytostatic activities against HeLa cells were affected by compounds 5a, 5c, 5e and 5k on MCF-7 cells by 5c, 5e and 5k and MIA PACa-2 cells by 5c and 5k. Differential effects at micromolar concentrations were observed for compounds 5a, 5c, 5e, 5k and 5l in Hep G2 cells, for compounds 5c, 5e, 5k and 5l in PC-3 cells and for compounds 5e, 5k and 5l in SW 620 cells.

Reactions of in situ generated N-Boc nitrones with aromatic and heteroaromatic grignard reagents: application to the synthesis of zileuton.

A new class of alpha-aromatic-N-hydroxylamines has been prepared by reaction of tert-butyl (phenylsulfonyl)alkyl-N-hydroxycarbamates with aromatic and heteroaromatic Grignard reagents. Reactions proceed via a base-assisted elimination of the phenylsulfonyl group leading to N-Boc nitrones. This methodology has been applied to the synthesis of zileuton.

Peptidyl 3-substituted 1-hydroxyureas as isosteric analogues of succinylhydroxamate MMP inhibitors.

To evaluate N-hydroxyurea as zinc binding group in the design of MMP inhibitors, two peptidyl 1-hydroxyureas were prepared by N-hydroxycarbamoylation of the diastereomeric dipeptides H-Leu-Phe-NHMe and H-D-Leu-Phe-NHMe. Peptidyl 1-hydroxyureas were more potent than the parent peptides, but dramatically weaker (4-5 orders of magnitude) than the isosteric (R)-succinylhydroxamate analogue, which displays IC(50) in the range of nM vs MMP-1, -3, -7 and sub-nM vs MMP-2, -8, and -9. The peptidyl 1-hydroxyurea 1a attained an IC(50) of 20 microM vs MMP-9, and substantially approaches inhibition of known N-hydroxyureas based on aminoacids or peptides against other zinc metalloenzymes and non-peptidic N-hydroxyureas against MMPs. Strong preference of the O-N1-C=O unit for the antiperiplanar amide bond conformation seems to be the major limit for more effective zinc chelation. Methylation of a peptidyl 1-hydroxyurea at N3, to promote the synperiplanar O-N1-C=O conformation required for zinc chelation and improve affinity, resulted in release of a methylimidazolidine-2,4-dione through an undesired intramolecular reaction reminiscent of the Edman peptide degradation.

Unnatural amino acid-substituted (hydroxyethyl)urea peptidomimetics inhibit gamma-secretase and promote the neuronal differentiation of neuroblastoma cells.

Gamma-secretase, exhibiting characteristics of aspartyl protease, mediates the intramembranous proteolysis of beta-amyloid precursor protein (APP) and Notch, and it is considered to be a prime pharmacological target in the development of therapeutics for Alzheimer's disease (AD). To identify compounds that block gamma-secretase-mediated proteolysis, we used a highly sensitive cell-based reporter gene assay for gamma-secretase in which Gal4/VP16-tagged C99-APP was expressed as the immediate substrate of gamma-secretase, and Gal4/VP16-tagged APP intracellular domain released by the gamma-secretase cleavage then activated the expression of the Gal4-driven luciferase reporter gene. Using this reporter assay, we demonstrated that the newly synthesized (hydroxyethyl)urea peptidomimetics, which contain unnatural amino acid moieties at positions P1' and/or P3', can effectively inhibit gamma-secretase activity and significantly reduce Abeta production. The gamma-secretase-dependent S3 cleavage of Notch was also consistently blocked by these (hydroxyethyl)ureas as evidenced by the decreased generation of the Notch intracellular domain, a prerequisite for the activation of Notch signaling. The inhibition of Notch signaling by active Jia compounds efficiently promotes the neuronal differentiation of neuroblastoma cells, intervening in tumorigenesis and the malignancy of neuroblastomas. Our results suggest that (hydroxyethyl)urea peptidomimetics containing unnatural amino acid substitutions could represent a novel class of gamma-secretase inhibitors with enhanced stability, providing the basis for the further development of effective therapeutics for AD and neuroblastomas.

Synthesis, aerobic cytotoxicity, and radiosensitizing activity of novel 2,4-dinitrophenylamine tethered 5-fluorouracil and hydroxyurea.

Two novel dual functional agents, 3[3-(2,4-dinitro-phenylamino)-propyl]-5-fluoro-1H-pyrimidine-2,4-dione 7 and N-[3-(2,4-dinitro-phenylamino)-propoxy]urea 8, resulting from linkage of 2,4-dinitrophenylamine through three carbon atoms with 5-fluorouracil 5 and hydroxyurea 6, respectively, were prepared and their in vitro aerobic cytotoxicities in HT-29 cell line with and without radiation were determined. Compounds 7 and 8 unlike their components were not cytotoxic but showed radiosensitizing activity.

Synthesis and X-ray crystal structure study of the hydroxyurea and hydantoin derivatives of L-valine.

The novel hydroxyurea 5 derivative of L-valine was prepared by aminolysis of N-(1-benzotriazolecarbonyl)-L-valine cyclohexanemethylamide 4 with hydroxylamine. The corresponding hydantoin derivative 6 was synthesized by base catalyzed cyclization of the amide 4. The exact stereostructure of hydantoin derivative 6 has been determined by X-ray crystal structure analysis. The chiral atom of the hydantoin ring in 6 has S configuration what is in agreement with its configuration in the starting L-valine. The molecules of 6 are joined into infinite chains by N-H...O intermolecular hydrogen bond. The infinite chains are additionally linked by two C-H...O hydrogen bonds, thus forming two-dimensional network. The hydantoin derivative of L-valine 6 and its L-leucine analogue LH have similar packing arrangements, so they are homostructural.

The novel L- and D-amino acid derivatives of hydroxyurea and hydantoins: synthesis, X-ray crystal structure study, and cytostatic and antiviral activity evaluations.

The novel L- and D-amino acid derivatives of hydroxyurea 5a-o were prepared by aminolysis of N-(1-benzotriazolecarbonyl)amino acid amides 4a-o with hydroxylamine. The hydantoin derivatives 6a-e,m,p were synthesized by base-catalyzed cyclization of amides 4, common precursors for 5 and 6. X-ray crystal structure analysis shows that the C5 atom in 6e possesses the S configuration, which is consistent with the configuration of the starting reagent, l-leucine. Among L-amino acid derivatives of hydroxyurea, 5h and 5i inhibited specifically murine leukemia and human T-lymphocytes (IC(50) = 10-19 microM) and showed selectivity with respect to normal human fibroblasts (WI 38). d-Amino acid derivatives of hydroxyurea 5m and 5o inhibited the growth of all tumor cell lines (IC(50) = 4.8-83.9 microM), but not the growth of normal fibroblasts (WI 38; IC(50) > 100 microM). Results on antiviral evaluations showed that N-(1-benzotriazolecarbonyl)amino acid amide 4m and hydantoin 6m had marked activity against the Davis strain of CMV (4m, EC(50) = 3.2 microg/mL; 6m, EC(50) = 4.0 microg/mL). However, these compounds showed also rather expressed cytotoxicity (4m, CC(50) = 43.4 microg/mL; 6m, CC(50) = 12.5 microg/mL(-1)).

Probing pockets S2-S4' of the gamma-secretase active site with (hydroxyethyl)urea peptidomimetics.

(Hydroxyethyl)urea peptidomimetics are potent inhibitors of gamma-secretase that are accessible in a few synthetic steps. Systematic alteration of P2-P4' revealed that the corresponding S2-S4' active site pockets accommodate a variety of substituents, consistent with the fact that this protease cleaves a variety of single-pass membrane proteins; however, phenylalanine is not well tolerated at P2'. A compound spanning P2-P3' was identified as a low nM inhibitor of gamma-secretase activity both in cells and under cell-free conditions.

Hydroxyurea analogues as kinetic and mechanistic probes of the nitric oxide producing reactions of hydroxyurea and oxyhemoglobin.

Derivatives of N-hydroxyurea that contain an N-hydroxy group react with oxyhemoglobin to form methemoglobin and variable amounts of nitrite/nitrate. Compounds with an unsubstituted -NHOH group produce the most nitrite/nitrate, which provides evidence for nitric oxide formation. The rate of reaction of these N-hydroxyurea derivatives with oxyhemoglobin correlates well with that compound's oxidation potential. Aromatic N-hydroxyureas react 25-80-fold faster with oxyhemoglobin than with N-hydroxyurea, suggesting other N-hydroxyurea analogues may be superior nitric oxide donors. Electron paramagnetic resonance spectroscopy shows that the formation of a low-spin methemoglobin-hydroxyurea complex is critical for iron nitrosyl hemoglobin formation. These results show that iron nitrosyl hemoglobin formation from the reaction of hydroxyureas and hemoglobin requires an unsubstituted -NHOH group and that the nitrogen atom of the non-N-hydroxy group must contain at least a single hydrogen atom. These results should guide the development of new hydroxyurea-based nitric oxide donors and sickle cell disease therapies.

Vilsmeier formylation of 2-carboxyindoles and preparation of O-benzylhydroxyureas on solid phase.

The Vilsmeier formylation has been introduced for the solid-phase functionalization of five different 2-carboxyindoles. The aldehyde functionality has been utilized in the preparation of O-benzylhydroxyureas.

N-(Hydroxyaminocarbonyl)phenylalanine: a novel class of inhibitor for carboxypeptidase A.

N-(Hydroxyaminocarbonyl)phenylalanine (1) was designed rationally as a new type of inhibitor for carboxypeptidase A (CPA). The designed inhibitor was readily prepared from phenylalnine benzyl ester in two steps and evaluated to find that rac-1 inhibits CPA in a competitive fashion with the Ki value of 2.09 microM. Surprisingly, inhibitor 1 having the D-configuration is more potent (Ki = 1.54 microM) than its antipode by about 3-fold. A possible explanation for the stereochemistry observed in the inhibition of CPA with 1 is presented.

New NO donors with antithrombotic and vasodilating activities, Part 28. N-(1-cyanoalkyl)-N-hydroxyureas.

Nineteen N-(1-cyanoalkyl)-N-hydroxyureas comprising aliphatic (3a-i, 4a, b, and 5a) and aromatic (3j-n, 4c, 5b) compounds were prepared, fourteen of them for the first time, and tested for antithrombotic (p.o. administration to rats, 60 mg/kg) effects. The N-(1-cyanocyclohexyl)-N-hydroxy-N'-phenylurea (3j) was most potent and inhibited laser-induced (35 mW, 50 ms) thrombus formation in arterioles by 21% and that in venules by 15%. The compounds form nitric oxide in vitro by the addition of a Fe3(+)-porphyrin complex and an oxygen donor. Moreover, the most active compound 3j in vivo exhibits the highest NO formation in vitro. Furthermore, it was shown that the cyano group is essential for the desired activities and NO formation. These results suggest that the title compounds act as NO donors.

(R)-(+)-N-[3-[5-[(4-fluorophenyl)methyl]-2-thienyl]-1-methyl- 2-propynyl]-N-hydroxyurea (ABT-761), a second-generation 5-lipoxygenase inhibitor.

Structure-activity optimization of inhibitory potency and duration of action of N-hydroxyurea containing 5-lipoxygenase inhibitors was conducted. The lipophilic heteroaryl template and the link group connecting the template to the N-hydroxyurea pharmacophore were modified. Inhibition of 5-lipoxygenase was evaluated in vitro in a human whole blood assay. An in vitro assay using liver microsomes from monkey was used to evaluate congeners for comparative rates of glucuronidation. (3-Heteroaryl-1-methyl-2-propynyl)-N-hydroxyureas were found to be more resistant to in vitro glucuronidation. The promising inhibitor N-[3-[5-(4-fluorophenoxy)-2-furyl]-1-methyl-2-propynyl]-N- hydroxyurea (6) was found to have stereoselective glucuronidation in monkey and man. The R enantiomer 7 provided longer duration of inhibition as evaluated by an ex vivo whole blood assay. Further optimization of the lipophilic template led to the discovery of (R)-(+)-N-[3-[5-[(4-fluorophenyl)methyl]-2- thienyl]-1-methyl-2-propynyl]-N-hydroxyurea (11) with more effective and prolonged inhibition of leukotriene biosynthesis than zileuton (1) and 7 in monkey and man. The optimized 5-lipoxygenase inhibitor 11 was selected for development as an investigational drug for leukotriene-mediated disorders.

Mutagenicity of the anticancer drug, caracemide, and related compounds for salmonella.

Caracemide, MeCON(CONHMe)(OCONHMe) (I), is a novel anticancer drug. Since it was derived from acetohydroxamic acid (II), a known mutagen, its potential metabolites and related compounds were synthesized and tested for mutagenicities in S. typhimurium TA98 and TA100. These compounds were: MeNHCONH(OCONHMe) (III), MeCONH(OCONHMe) (IV), MeCONOH(CONHMe) (V), MeNHCOONH2 X HCl (VI), MeNHCONHOH (VII), MeNHCOON(CONHMe)2 (VIII), and NOH(CONHMe)2 (IX). The mutagenicities in the absence of rat liver homogenate were: (VI) much greater than (IV) greater than (II), (III), (V). The other compounds were not mutagenic. (I) was mutagenic only in the presence of rat liver homogenate. The doses required to demonstrate mutagenicities of these compounds were from 0.05 to 5 mumoles/plate. The major hydrolytic products at 25 degrees C, pH 7, were (III), (IV), and (V) from (I); (II) and (III) from (IV); and (II), (III), (VII) and MeNHCONH(OCOMe) (X) from (V). (III) was stable at pH 7. Treatment of (IV) with HCl yielded (VI). Hydrolysis of (III) or (V) with ammonia yielded (VII). These results suggest that caracemide may be activated enzymatically or nonenzymatically by deacetylation or decarbamoylation, and its anticancer activity may be related to the reactivity of its metabolites with DNA. The synthetic procedures and characterizations of new compounds (IV), (V) and (X) are described.