In vivo characterization of hydroxamic acid inhibitors of 5-lipoxygenase

The hydroxamic acid functionally can be incorporated into simple molecules to produce potent inhibitors of 5-lipoxygenase. The ability of many of these hydroxamates to inhibit leukotriene synthesis in vivo has been measured directly with a rat peritoneal anaphylaxis model. Despite their potent enzyme inhibitory activity in vitro, many orally dosed hydroxamic acids only weakly inhibited leukotriene synthesis in vivo. This discrepancy is attributable at least in part to the rapid metabolism of hydroxamates to the corresponding carboxylic acids, which are inactive against the enzyme. A study of the structural features that affect this metabolism revealed that 2-arylpropionohydroxamic acids are relatively resistant to metabolic hydrolysis. Several members of this class of hydroxamates are described that are orally active inhibitors of leukotriene synthesis.     

The synthesis of potential 5-lipoxygenase inhibitors. 1

A Synthetic Route to Potential Inhibitors of 5-Lipoxygenase, I Derivatives of enamino;(cyano)pyrrol are able to inhibit 5-lipoxygenase. Bulky substituents at position 2 of an aromatic moiety seem to increase the inhibitory potency. As a model compound, a 2,4-dinitrophenyl derivative was synthesized, and a new synthetic route was developed for enaminopyrrols with sterically demanding substituents.     

Novel 1,2,4-oxadiazoles and 1,2,4-thiadiazoles as dual 5-lipoxygenase and cyclooxygenase inhibitors.

A series of 1,2,4-oxadiazoles and 1,2,4-thiadiazoles containing a 2,6-di-tert-butylphenol substituent were prepared and evaluated as dual inhibitors of 5-lipoxygenase and cyclooxygenase in rat basophilic leukemia (RBL-1) cells. Several of these compounds show oral efficacy in the rat carrageenan footpad edema (CFE) and mycobacterium footpad edema (MFE) antiinflammatory models, without concomitant gastric ulceration. Structure-activity relationships are discussed. The best compounds (ID40 values in MFE of 3-8 mg/kg po) contain guanidine-derived substituents on the heterocyclic ring.     

Synthesis of acyclonucleoside hydroxamic acids as inhibitors of ribonucleotide reductase

N-Hydroxy-alpha-(2-hydroxyethoxy)-1(2H)-pyrimidineacetamides 1-3 were synthesized as potential antitumor agents whose mechanism of action would involve inhibition of ribonucleoside diphosphate reductase (RDPR, EC 1.17.4.1). Acyclonucleoside esters 6-8 were prepared by the stannic chloride catalyzed reaction of methyl chloro[2-(phenylmethoxy)ethoxy]acetate (5) with various silylated pyrimidines, generated in situ from the bases and bis(trimethylsilyl)acetamide. Catalytic didebenzylation of hydroxamate 11 gave 1, while 2 and 3 were synthesized by the reaction of lactones 14 and 22, respectively, with hydroxylamine. In vitro acyclonucleoside hydroxamic acids 1-3 were 3-10-fold less potent than hydroxyurea against calf thymus cytidine diphosphate reductase. 5-Fluorouracil derivative 2 is nearly equipotent with hydroxyurea in inhibiting the growth of HeLa cells, while 1 is a much weaker inhibitor and cytidine derivative 3 is devoid of activity at 200 micrograms/mL.     

5-lipoxygenase: properties, pharmacology, and the quinolinyl(bridged)aryl class of inhibitors.

In conclusion, an effective modulator of the AA cascade for the treatment of asthma and other inflammatory diseases may require 5-LO inhibitory activity as well as LTD4 antagonism in order to limit the effects of LTB4, LTD4, and 5-HPETE. The unknown role of LTC4 with respect to bronchoconstriction and mucus production could mask the efficacy of a pure LTD4 antagonist in man, whereas the chemotactic property of LTB4 for eosinophils can contribute to lung inflammation. Indeed, it is observed that the blood of patients with bronchial asthma has increased numbers of hypodense eosinophils. In addition, the formation of lipid-derived peroxide radicals, such as 5-HPETE, are believed to be responsible for various types of cellular injuries associated with the inflammatory disease process. Because inhibition of the CO pathway is thought to explain the therapeutic effects of nonsteroidal antiinflammatory agents in rheumatic diseases, a 5-LO inhibitor with CO inhibitory activity may also be desirable profile for an antiasthma agent. The validation of the LT hypothesis of disease had to wait for the demonstration of a clinical effect by either a LTD4 receptor antagonist or a LT synthesis inhibitor (5-LO inhibitor). Only very recently has this evidence become available and it is now apparent that compounds that antagonize LTD4 receptors or inhibit LT synthesis have shown clinical efficacy in a wide range of diseases. Due to the breakthrough nature of this approach, certain of these compounds are being considered for expedited development. The absence of side effects seen in the clinical trials of selective 5-LO inhibitors is gratifying and argues that LTs are not important in homeostasis. Only time will tell whether 5-LO inhibitors will take their place in the therapeutic armamentarium; however, the recent demonstration of clinical efficacy by a number of these compounds is a significant step in this direction.     

Are tolfenamic acid and tenidap dual inhibitors of 5-lipoxygenase and cyclo-oxygenase?

Tolfenamic acid and tenidap have been reported to be dual inhibitors of cyclo-oxygenase and 5-lipoxygenase. In this study inhibition of 5-lipoxygenase by tenidap and tolfenamic acid in plasma-free leukocyte suspensions (IC50 values = 10 microM) required concentrations more than 100 fold higher than those which inhibited cyclo-oxygenase (IC50 values = 0.05 and 0.02 microM respectively). The potencies of tolfenamic acid and tenidap as cyclo-oxygenase inhibitors were markedly reduced in blood (IC50 = 6.5 and 10 microM respectively) and neither significantly inhibited 5-lipoxygenase. Since both drugs also failed to inhibit 5-lipoxygenase in rat blood ex vivo, we conclude that, at physiological levels of plasma proteins, tolfenamic acid and tenidap are selective cyclo-oxygenase inhibitors.     

Immunoregulatory properties of novel specific inhibitors of 5-lipoxygenase

The immunoregulatory properties of novel specific inhibitors of 5-lipoxygenase (5-LO) were investigated in vitro. These novel acetohydroxamic acids were found to inhibit the proliferation of lymphocytes in response to Interleukin-1 (IL-1) and Interleukin-2 (IL-2). Mitogen- and antigen-induced lymphoproliferation were likewise inhibited and so too was the spontaneous proliferation of transformed cells of myeloid and lymphoid origin. In this respect the compounds were one-tenth as potent as azathioprine on a molar basis. The production of IL-1 and IL-2 remained unaffected. Inhibition of lymphocyte proliferation was not related to inhibition of 5-LO. The latter occurred at 40-fold lower concentrations of 5-LO inhibitor. Moreover, inhibition of lymphoproliferation was not reversible in the presence of excess arachidonic acid. IL-1-induced collagenase production by chondrocytes was unaffected by 5-LO inhibitors. Contrary to a number of published reports, products of 5-LO do not appear to be obligatory mediators in IL-1 and IL-2 signal transduction. The immunosuppressive action of the 5-LO inhibitors described here seems to be a novel secondary property of the compounds.     

Benzothiazole hydroxy ureas as inhibitors of 5-lipoxygenase: use of the hydroxyurea moiety as a replacement for hydroxamic acid.

A novel series of N-[(2-benzothiazolylthio)alkyl]-N'-hydroxyurea derivatives (9-25) was synthesized and evaluated for biological activity as inhibitors of 5-lipoxygenase both in vivo (mouse zymosan peritonitis assay) and in vitro (Ca2+ ionophore-stimulated human peripheral blood leukocyte model). The compounds of this series were based on the corresponding hydroxamic acid derivatives (1, 3, 4, and 5) which were moderately active in vitro but inactive in vivo. A number of compounds in the hydroxyurea series exhibited oral activity for 5-lipoxygenase inhibition. Results of studies relating structure to in vivo and in vitro 5-lipoxygenase activity are reported.     

Hyperforin is a dual inhibitor of cyclooxygenase-1 and 5-lipoxygenase

The acylphloroglucinol derivative hyperforin is the major lipophilic constituent in the herb Hypericum perforatum (St. John's wort). The aim of the present study was to investigate if hyperforin as well as extracts of H. perforatum can suppresses the activities of 5-lipoxygenase (5-LO) and cyclooxygenases (COX), key enzymes in the formation of proinflammatory eicosanoids from arachidonic acid (AA). In freshly isolated human polymorphonuclear leukocytes stimulated with Ca(2+) ionophore A23187, hyperforin inhibited 5-LO product formation with IC(50) values of about 1-2 microM, in the absence or presence of exogenous AA (20 microM), respectively, being almost equipotent to the well-documented 5-LO inhibitor zileuton (IC(50) = 0.5-1 microM). Experiments with purified human 5-LO demonstrate that hyperforin is a direct 5-LO inhibitor (IC(50) approximately 90 nM), acting in an uncompetitive fashion. In thrombin- or ionophore-stimulated human platelets, hyperforin suppressed COX-1 product (12(S)-hydroxyheptadecatrienoic acid) formation with an IC(50) of 0.3 and 3 microM, respectively, being about 3- to 18-fold more potent than aspirin. At similar concentrations, hyperforin suppressed COX-1 activity in platelets in presence of exogenous AA (20 microM) as well as in cell-free systems. Hyperforin could not interfere with COX-2 product formation and did not significantly inhibit 12- or 15-LO in platelets or leukocytes, respectively. We conclude that hyperforin acts as a dual inhibitor of 5-LO and COX-1 in intact cells as well as on the catalytic activity of the crude enzymes, suggesting therapeutic potential in inflammatory and allergic diseases connected to eicosanoids.     

1,4-Dihydronaphthoquinones, hydroindoloquinones, benzofurans, and benzothiophenes as inhibitors of 5-lipoxygenase. Synthesis and structure-activity studies

A series of substituted 1,4-dihydronaphthoquinones, hydroindoloquinones, benzofuran-4,7-dihydroquinones, and benzothiophene-4,7-dihydroquinones were synthesized and evaluated for inhibitory activity against 5-lipoxygenase. These compounds were found to be active in vitro for LTC4/D4 inhibition with the potencies (IC50's) ranging from 0.2 to 85 microM. Active 1,4-dihydronaphthoquinone acetates (IC50 less than 20 microM) were evaluated in an ex vivo LTB4 inhibition assay. The acetates of 1,4-dihydronaphthoquinones containing the alkyl substituent(s) (2-n-butyl, 11, and 2,3-diethyl, 15) exhibited the best activity in LTC4/D4 inhibition (IC50 = 0.2-0.4 microM, in vitro) as well as in LTB4 inhibition (60-75% inhibition).     

Synthesis and biological evaluation of N-aryl-4-aryl-1,3-thiazole-2-amine derivatives as direct 5-lipoxygenase inhibitors

Biological evaluation of N-aryl-4-aryl-1,3-thiazole-2-amine derivatives was examined for anti-inflammatory activity in in vitro and in vivo assays. The thiazole compounds showed direct inhibition of 5-lipoxygenase (LOX) that is a key enzyme of leukotrienes synthesis and involved in the inflammation-related diseases, including asthma and rheumatoid arthritis. To optimize biological activity, we synthesized 1,3-thiazole-2-amine derivatives and investigated for structure and activity relationship. Especially, N-(3,5-dimethylphenyl)-4-(4-chlorophenyl)-1,3-thiazole-2-amine was shown to have a potent anti-inflammatory activity as a 5-LOX inhibitor.     

Styrylpyrazoles, styrylisoxazoles, and styrylisothiazoles. Novel 5-lipoxygenase and cyclooxygenase inhibitors.

A series of styrylpyrazoles, styrylisoxazoles, and styrylisothiazoles were prepared and found to be dual inhibitors of 5-lipoxygenase and cyclooxygenase in rat basophilic leukemia cells. Compounds from this series also were found to inhibit the in vivo production of LTB4 when dosed orally in rats. Among these compounds, di-tert-butylphenols 19 and 33 exhibit oral activity in various models of inflammation and, most importantly, are devoid of ulcerogenic potential.     

邻硝基苯丙酸取代的异羟肟酸类组蛋白去乙酰化酶抑制剂的设计、合成与生物活性研究

目的合成一系列新型异羟肟酸类组蛋白去乙酰化酶(HDACs)抑制剂,并对这些化合物做了活性评价。方法以硝基苯衍生物,对位有取代的苯甲酰氯,邻硝基苯丙酸为原料合成这一系列化合物。MTT法检测化合物对A549、BEL-7402、MGC-803的抑制作用,并通过HDACs抑制实验进一步验证化合物活性,对抑制活性好的化合物进一步用双染法检测化合物诱导细胞凋亡情况,并通过急性毒性实验测定化合物的毒性。结果化合物7d、7f、7h、7j对A549细胞的抑制作用优于阳性对照SAHA。通过对HDACs抑制实验,化合物7d、7f、7h、7j均具有较好的酶抑制作用,与MTT实验结果基本一致。结论此类新型异羟肟酸类HDACs抑制剂能有效抑制HDACs,并降低细胞毒性,有较为广阔的研究前景,值得进一步研究。     

Synthesis and antiallergic activity of a novel series of 5-lipoxygenase inhibitors

A series of novel substituted [[(phenoxymethyl)phenyl]amino]oxoalkanoic acid esters have been synthesized. These compounds were tested in vitro for their ability to inhibit the synthesis of 5-hydroxyeicosatetraenoic acid and leukotriene (LT) B4 from rat polymorphonuclear leukocytes (PMN) and in vivo as inhibitors ovalbumin- (OA) and LTD4-induced bronchospasm in the guinea pig. Compounds 5-12 and 25 had IC50's between 1 and 5.6 microM in the rat PMN 5-lipoxygenase assay. Compounds 1, 3, and 16 inhibited OA-induced bronchoconstriction (61%, 64%, and 57%, respectively), but only 1 showed activity against LTD4-induced bronchoconstriction. When tested against LTD4-induced contraction of isolated guinea pig tracheal spiral strips, 1 was a competitive inhibitor with a pKB of 4.94.     

Guinea pig whole blood 5-lipoxygenase assay: utility in the assessment of potential 5-lipoxygenase inhibitors.

Guinea pigs are widely used in the study of the role of leukotrienes in airway pathophysiology. Extensive research efforts have utilized this species in the development of potential therapeutic agents associated with inhibition of leukotriene production (e.g. 5-lipoxygenase inhibitors and 5-lipoxygenase-activating protein antagonists) for the treatment of acute bronchospasm in asthma. We now report, for the first time, an ex vivo whole blood 5-lipoxygenase assay in guinea pigs which should prove useful in the future development of leukotriene biosynthesis inhibitors. Addition of 150 microM arachidonic acid (AA) to heparinized whole blood for 5 min prior to the stimulation with 20 micrograms/mL A23187 resulted in a 10-fold increase in leukotriene B4 (LTB4; 11.36 +/- 1.55 ng/mL) above basal (0.96 +/- 0.29 ng/mL) within 10 min. To further validate the utility of the assay, we utilized the 5-lipoxygenase inhibitor BW A4C. Pretreatment of guinea pig whole blood with BW A4C in vitro prior to stimulation resulted in a concentration-dependent inhibition of LTB4 production (IC50 = 229 nM), whereas thromboxane B2 (TXB2) production was unaffected. Likewise, when BW A4C was administered to guinea pigs intravenously (3 mg/kg), we observed a rapid and marked (approximately 90%) reduction in whole blood LTB4 production which returned to control (pre-drug values) by 5 hr. In contrast, TXB2 production was unaffected over the same experimental time period. In summary, we have described a whole blood assay which can discriminate 5-lipoxygenase inhibitors both in vitro and in vivo. Furthermore, this assay system will be of use in determining the potency, efficacy, selectivity, and pharmacodynamic properties of 5-lipoxygenase inhibitors in guinea pigs.