A. I. Vogel, Preparatyka organiczna (WNT Warszawa,), p. Zielińska J., Makowski M., Maj K., Liwo A., Chmurzyński L.() Anal. A.I. Vogel. Preparatyka Organiczna, WNT, Warszawa (), p. F.V. Lovecchio, E.S. Gore, D.H. Bush. J. Am. Chem. Soc., 96 (), p. Soc. (), p. A.I. Vogel. Preparatyka Organiczna W.N.T. Warszawa ( ), p. C.G. Hatchard, C.A. Parker. Proc. Roy. Soc., A (), p.
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This application claims the benefit of International Application No. The sequence listing is identified on the electronically filed text file as follows:. File Name Date of Creation Size bytes seqlist.
The present invention relates generally to phosphatidylinositol 3-kinase PI3K enzymes, and more particularly to selective inhibitors of PI3K activity and methods of using such inhibitors. The enzyme responsible for generating these phosphorylated signaling products is phosphatidylinositol 3-kinase PI 3-kinase; PI3K.
Levels of phosphatidylinositol-3,4,5-triphosphate PIP3the primary product of PI 3-kinase activation, increase upon treatment of cells with a variety of agonists. PI 3-kinase activation, therefore, is believed to be involved in a range of cellular responses including cell growth, differentiation, and apoptosis Parker et al. Though the downstream targets of phosphorylated lipids generated following PI 3-kinase activation have not been well characterized, emerging evidence suggests that pleckstrin-homology domain- and FYVE-finger domain-containing proteins are activated when binding to prepxratyka phosphatidylinositol lipids Sternmark et al.
Presently, the PI 3-kinase enzyme family is divided into three classes based on their substrate specificities. Class I PI3Ks can phosphorylate phosphatidylinositol PIphosphatidylinositolphosphate, and phosphatidylinositol-4,5-biphosphate PIP2 to produce phosphatidylinositolphosphate PIPphosphatidylinositol-3,4-biphosphate, and phosphatidylinositol-3,4,5-triphosphate, respectively.
The initial purification and molecular cloning of PI 3-kinase revealed preparatka it was a heterodimer consisting of p85 and p subunits Otsu et al.
Preparatyka organiczna vogel pdf download
More specifically, three of the catalytic subunits, i. As described below, the patterns of expression of each of these PI3Ks in human cells and tissues also are distinct. Though a wealth of information has been accumulated on the cellular functions of PI 3-kinases in general, the roles played by the individual isoforms are largely unknown. This protein was identified as related to the Saccharomyces cerevisiae protein: Vps34p, a protein involved in vacuolar protein processing.
See Hiles et al. It is expressed at high levels in lymphocytes and lymphoid tissues, suggesting that the protein might play a role in PI 3-kinase-mediated signaling in the immune system. See also, Vanhaesebroeck et al.
In addition, prepaartyka binding of Ras proteins also upregulates PI 3-kinase activity. The p regulatory subunit for PI3 Kgamma was originally cloned in swine, and the human ortholog identified subsequently Krugmann et al.
This organicznaa discloses preparation of a chimeric fusion protein in a.ivogel a residue fragment of p85 known as the inter-SH2 iSH2 region is fused through a linker region to the N-terminus of murine p Thus, PI 3-kinases can be defined by their amino acid identity or by their activity.
See generally, Hunter, Cell, PI 3-kinase also appears to be involved in a number of aspects of leukocyte activation. A passociated PI 3-kinase activity has been shown to physically associate with the cytoplasmic domain of CD28, which is an important costimulatory molecule for the activation of T-cells in response to antigen Pages et al.
Activation of T cells through CD28 lowers the threshold for activation by antigen and increases the magnitude and duration of the proliferative response. These effects are linked to increases in the transcription of a number of genes including interleukin-2 IL2an important T cell growth factor Fraser et al.
Mutation of CD28 such that it can no longer preparatka with PI 3-kinase leads to a failure to initiate IL2 production, suggesting a critical role for PI 3-kinase in T cell activation.
Specific inhibitors against individual members of a family of enzymes provide invaluable tools for deciphering functions of each enzyme. Two compounds, LY and wortmannin, have been widely used as PI 3-kinase inhibitors. Hence, the utility of these compounds in studying the roles of individual Class I PI 3-kinases is limited. Based on studies using wortmannin, evidence exists that PI 3-kinase function also is required for some aspects of leukocyte signaling through G-protein coupled receptors Thelen et al.
Moreover, it has been shown that wortmannin and LY block neutrophil migration and superoxide release. However, because these compounds do not distinguish among the various isoforms of PI3K, it remains unclear which particular PI3K isoform or isoforms are involved in these phenomena.
In view of the above considerations, it is clear that existing knowledge is lacking with respect to structural and functional features of the PI 3-kinase enzymes, including subcellular localization, activation states, substrate affinities, and the like. Moreover, the functions that these enzymes perform in prsparatyka normal and diseased tissues remains to be elucidated. The coexpression in these tissues of other PI3K isoforms has heretofore confounded efforts to segregate the activities of each enzyme.
Furthermore, separation of the activities of the various PI3K isozymes may not be possible without identification of inhibitors that demonstrate selective inhibition characteristics. Indeed, a.i.votel presently preparztyka not aware that such selective, or better, specific, inhibitors of PI3K isozymes have been demonstrated.
A need also remains for selective or specific inhibitors of PI3K isozymes, such that the functions of each isozyme can be better characterized. The leukocytes can comprise cells selected from the group consisting of neutrophils, B lymphocytes, T lymphocytes, and basophils. For example, in cases wherein the leukocytes comprise neutrophils, the method comprises disrupting at least one neutrophil function selected from the group consisting of stimulated superoxide release, stimulated exocytosis, and chemotactic migration.
Preferably, the method does not substantially disrupt bacterial phagocytosis or bacterial killing by the neutrophils. In cases pgeparatyka the leukocytes comprise B lymphocytes, the method comprises disrupting proliferation of the B lymphocytes or antibody production by the B lymphocytes.
In cases wherein the leukocytes comprise T lymphocytes, the method comprises disrupting proliferation of the T lymphocytes. In cases wherein the leukocytes comprise basophils, the method comprises disrupting histamine release by the basophils. R 2 and R 3independently, are hydrogen, organicznz, or C alkyl. R 7 is hydrogen or R 5 and R 7 are taken together with the atoms to which they are attached to form a five- or six-membered saturated ring.
Another aspect of the present invention is to provide a method of treating a medical condition mediated by neutrophils comprising administering prpearatyka a mammal in need thereof a therapeutically effective amount of a compound of structural formulae I or II. Exemplary medical conditions that can be treated according to the method include those conditions characterized by an undesirable neutrophil function selected from the group consisting of stimulated superoxide release, stimulated exocytosis, and chemotactic migration.
Preferably, according to the method, phagocytic activity or bacterial killing by neutrophils is substantially uninhibited. Still another preparatgka of the present invention is to provide preparatykq method of disrupting a function of osteoclasts comprising contacting osteoclasts with a compound of structural formulae I or II. Another aspect of the present invention is to provide a method of ameliorating preparattyka bone-resorption organoczna in a mammal in need thereof comprising administering to the mammal a therapeutically effective amount of a compound of structural formulae I or II.
A preferred bone-resorption disorder amenable to treatment according to the method is osteoporosis. Yet another aspect of the present invention is to provide a method of inhibiting the growth or proliferation of cancer cells of hematopoietic origin comprising contacting the cancer cells with a compound of a.i.bogel formulae I or II. The method can be advantageous in inhibiting the growth or proliferation of cancers a.i.ogel from the group consisting of lymphomas, multiple myelomas, and leukemias.
Still another aspect of the present invention is to provide a method of disrupting leukocyte function comprising contacting leukocytes with a compound of structural formulae I or II. Another aspect of the present invention is to provide an article of manufacture for human pharmaceutical use comprising:.
These and other aspects and advantages of the present invention will become apparent from the following detailed description of the preferred embodiments, which are provided to enhance the understanding of the invention without limiting the scope of the invention.
Orgzniczna methods include in a.i.vogel, in vivo, and ex vivo organicza. Other methods of the invention include enabling the further characterization of the physiological role of the isozyme. The cells can be in peeparatyka, i. Alternatively, the cells can be isolated as discrete cells or in a tissue, for ex vivo or in vitro methods.
Potency typically is expressed as the concentration of a compound required to achieve a certain result.
The greater the potency, the less compound required to perform its intended function. In vitro potency typically is expressed in terms of IC 50 values measured using a dose-response assay.
IC 50 values can be a.i.ovgel by contacting a sensitive assay system with a compound of interest over a range of concentrations, preparatykw concentrations at which no or minimal effect is observed, through higher concentrations at which partial effect is observed, to saturating concentrations at prepwratyka a maximum effect is observed. Theoretically, such assays of the dose-response effect of inhibitor compounds can be described as a sigmoidal curve expressing a degree of inhibition as a function of concentration when plotted on a log scale.
IC 50 values can be determined using either conventional biochemical acellular assay techniques or cell-based assay techniques well known to those of ordinary skill in the art. An example of such an assay is provided in the examples below. More preferably, the assay is repeated from 3 to 10 or more times, with the IC 50 value expressed as the mean of the values obtained. Still more preferably, the assay is performed a number of times sufficient to generate a statistically reliable mean IC 50 value, using statistical methods known to those of ordinary skill organjczna the art.
In other embodiments, the compounds of the invention exhibit IC 50 values from about 0. The most preferred compounds of the present invention, therefore, have a low IC 50 value vs. In this context, agents can be used therapeutically in vivo to retard or eliminate the a.i.vpgel of aberrantly replicating cells.
The agents also can be used in vivo as preparatyja prophylactic to prevent aberrant cell proliferation or the manifestation of symptoms associated therewith. Examples of ex vivo cell populations include cell cultures and biological samples such as fluid or tissue samples prrparatyka humans or animals.
Such samples can be obtained by methods well known in the art. Exemplary biological fluid samples include prepagatyka, cerebrospinal fluid, urine, saliva. Exemplary tissue samples include tumors and biopsies.
In this context, the present compounds can be in numerous applications, both therapeutic and experimental. In one embodiment, the present invention provides a method of inhibiting leukocyte function. More particularly, the present invention provides methods of inhibiting or suppressing functions of neutrophils and T and B lymphocytes. For example, it has been observed that the compounds of the present invention elicit inhibition of classical neutrophil functions, such as stimulated superoxide production, stimulated exocytosis, and chemotactic migration.
However, it further has been observed that a method of the present invention permits suppression of certain functions of neutrophils, while not substantially affecting other functions of these cells. Thus, the present invention includes methods of inhibiting neutrophil functions, without substantially inhibiting phagocytosis of bacteria. Such functions include, without limitation, stimulated superoxide release, stimulated exocytosis or degranulation, chemotactic migration, adhesion to vascular endothelium e.
The inflammatory functions of neutrophils can be distinguished from the bacterial killing functions exhibited by these cells, e. Accordingly, the present invention further includes methods of treating disease states in which one or more of the inflammatory functions of neutrophils are abnormal or undesirable.
Accordingly, the compounds of the invention advantageously permit treatment of conditions associated with an undesirable endogenous immune response stimulated by at least one endogenous factor without compromising the ability to fight infection.
Accordingly, the compounds of the invention advantageously permit treatment of individuals having an inflammatory condition where leukocytes are found to be accumulating at the site of insult or preparatka tissue. Thus, the present invention includes methods of using compounds of structural formulae I or II for inhibiting lymphocyte proliferation, or for prfparatyka antibody production by B lymphocytes.
Other methods enabled by the present invention include methods of treating disease states in which one or more of these perparatyka functions are abnormal or undesirable. The methods of this invention can be practiced using compounds of structural formulae I or II. The methods can be practiced using a racemic mixture of the compounds or oorganiczna specific enantiomer.
In preferred embodiments, the S-enantiomer of the compounds are utilized in the present methods. The methods can be practiced using, for example, the following compounds of the invention, organicana the invention is not limited to these compounds.
In particular, the methods of the invention can be practiced using compounds having the general structural formula Lrganiczna.