Biomedical subjects
Publications and source records attributed to P Wipf.
[formula: see text] A mild and highly efficient cyclization of beta-hydroxy amides to oxazolines is described using DAST and Deoxo-Fluor reagents. A one-pot protocol for the synthesis of oxazoles from beta-hydroxy amides is also presented.
The isolation of the cytotoxic Lissoclinum sp. metabolite trunkamide A was reported in 1996. After completion of a total synthesis in 1999, it became clear that the structure of this marine natural product had to be revised. We now report the first preparation of actual trunkamide A in a total synthesis that serves as an unambiguous structural and stereochemical proof. Highlights of our synthetic strategy are a Lewis acid assisted aziridine opening that was used for the preparation of the novel reverse-prenylated serine and threonine side chains as well as an efficient oxazoline-thiazoline interconversion on the macrocyclic skeleton. In addition, several stereoisomers prepared by complementary synthetic protocols serve to illustrate the general scope of our methodology and confirm the configurational assignment.
Phosphorylation of serine, threonine, and tyrosine controls fundamental mammalian cell events and is achieved by kinases which, in turn, are in dynamic relationship with phosphatases. Few selective inhibitors of protein tyrosine and dual specificity phosphatases are readily available. Based on SAR studies of naturally occurring phosphatase inhibitors and following up on previously published research, we have designed a new pharmacophore model V and synthesized a new library of functional analogues of V. All synthetic steps were carried out and optimized employing combinatorial chemistry methods on Wang resin. All compounds were tested in vitro for their ability to inhibit recombinant human protein tyrosine (PTP1B) and dual-specificity (Cdc25B(2) and VHR) phosphatases. Three of the approximately 70 compounds in our library inhibited Cdc25B(2) by 50% at 375-490 microM. No compounds inhibited PTP1B, and only one blocked VHR. Cell-culture studies revealed no toxicity to human breast cancer cells with two of the phosphatase inhibitors.
Both solid- and liquid-phase combinatorial chemistry have emerged as powerful tools for identifying pharmacologically active compounds and optimizing the biological activity of a lead compound. Complementary high-throughput in vitro assays are essential for compound evaluation. Cell-based assays that use optical endpoints permit investigation of a wide variety of functional properties of these compounds including specific intracellular biochemical pathways, protein-protein interactions, and the subcellular localization of targets. Integration of combinatorial chemistry with contemporary pharmacology now represents an important factor in drug discovery and development.
Dual specificity protein phosphatases (DSPases) are key regulators of signal transduction, oncogenesis and the cell cycle. Few potent or specific inhibitors of DSPases, however, are readily available for these pharmacological targets. We have used a combinatorial/parallel synthetic approach to rigidify the variable core region and modify the side chains of 4-(benzyl-(2-[2,5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)- 2-decanoylamino butyric acid (or SC-alphaalphadelta9), which is the most active element in a previously described library of phosphatase inhibitors (Rice, R. L.; Rusnak, J. M.; Yokokawa, F.; Yokokawa, S.; Messner, D. J.; Boynton, A. L.; Wipf, P.; Lazo, J. S. Biochemistry 1997, 36, 15965). Several analogues were identified as effective inhibitors of the protein tyrosine phosphatase (PTPase) PTP1B and the DSPases VHR and Cdc25B2. Two compounds, FY3-alphaalpha09 and FY21-alphaalpha09, were partial competitive inhibitors of Cdc25B2 with Ki values of 7.6+/-0.5 and 1.6+/-0.2 microM, respectively. FY21-alphaalpha09 possessed only moderate activity against PTP1B. Consistent with its in vitro anti-phosphatase activity, FY21-alphaalpha09 inhibited growth in MDA-MB-231 and MCF-7 human breast cancer cell lines. FY21-alphaalpha09 also inhibited the G2/M transition in tsFT210 cells, consistent with Cdc25B inhibition. Several architectural requirements for DSPase inhibition were revealed through modification of the side chain moieties or variable core region of the pharmacophore, which resulted in decreased compound potency. The structure of FY21-alphaalpha09 provides a useful platform from which additional potent and more highly selective phosphatase inhibitors might be generated.
We previously showed that SC-alphaalphadelta9 (4-(benzyl-(2-[(2, 5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylami no butyric acid) is a novel antiphosphatase agent that selectively inhibits the growth of transformed cells in culture and affects elements of insulin-like growth factor-1 (IGF-1) signaling. We now show that SC-alphaalphadelta9 induces IGF-1-resistant apoptosis and kills tumor cells in vivo. In cultured murine 32D cells, SC-alphaalphadelta9 induced concentration-dependent apoptosis that was blocked by ectopic Bcl-2 expression. No apoptosis was detected in 32D cells treated with the congener SC-alpha109, which lacks the ability to disrupt IGF-1 signaling. After interleukin-3 withdrawal or etoposide treatment, exogenous IGF-1 prevented apoptosis and elevated levels of Cdc2, a biochemical indicator of a functional IGF-1 receptor pathway. In contrast, exogenous IGF-1 did not prevent apoptosis or loss of Cdc2 expression caused by SC-alphaalphadelta9. Furthermore, IGF-1 receptor overexpression failed to protect cells against SC-alphaalphadelta9-induced apoptosis. Kinetic analyses demonstrated that Cdc2 down-regulation after SC-alphaalphadelta9 treatment preceded both apoptosis and loss of the IGF-1 receptor, indicating that loss of Cdc2 was a direct effect of SC-alphaalphadelta9 treatment and not secondary to cell death. IGF-1 receptor autophosphorylation studies indicated that SC-alphaalphadelta9 did not interact directly with the IGF-1 receptor nor bind to the growth factor itself, suggesting a site of action distal to the IGF-1 receptor. In the SCCVII murine tumor model, a single i.p. injection of SC-alphaalphadelta9 caused a dose-dependent decrease in clonogenic cell survival. The IC(50) of SC-alphaalphadelta9 was 35 mg/kg, comparable to 25 mg/kg carboplatin. The ability to induce IGF-1-resistant apoptosis distinguishes SC-alphaalphadelta9 from other apoptosis-inducing agents and suggests compounds of this class deserve further study as potential anticancer agents.
The Cdc25 dual specificity phosphatase family has a central role in controlling cell cycle progression and has been implicated in the etiology of cancer. One compound, 4-(benzyl-(2-[(2, 5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylami no butyric acid (SC-alpha alpha delta 9), was previously identified as the most potent reported synthetic inhibitor of Cdc25 phosphatases in vitro. In the present study, we demonstrate that SC-alpha alpha delta 9 inhibited Cdc25-dependent cell cycle progression at both G1 and G2/M phase using tsFT210 cells, which express a temperature-sensitive Cdc2 mutant. SC-alpha alpha delta 9 blocked both G2/M transition and dephosphorylation of Cdc2 in a concentration-dependent manner. SC-alpha alpha delta 9 also enhanced tyrosine phosphorylation of both Cdk2 and Cdk4, and decreased Cdk4 kinase activity. Both of the kinases are potent regulators of G1 transition. Furthermore, closely related chemical analogs that lacked Cdc25 inhibitory activity failed to block cell cycle progression at both G1 and G2/M, and did not affect Cdc2 phosphorylation or Cdk4 kinase activity. SC-alpha alpha delta 9 did not alter p53, p21 or p16 levels. Our results support the hypothesis that the disruption in cell cycle transition caused by SC-alpha alpha delta 9 was due to intracellular Cdc25 inhibition. We propose that the SC-alpha alpha delta 9 pharmacophore could be useful in further clarifying the role of Cdc25 phosphatase-dependent pathways in checkpoint control, oncogenesis, and apoptosis.
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Chiral molecules are characterized by a specific rotation angle, the angle through which plane-polarized light is rotated on passing through an enantiomerically enriched solution. Recent developments in methodology allow computation of both the sign and the magnitude of these rotation angles. However, a general strategy for assigning the individual contributions that atoms and functional groups make to the optical rotation angle and, more generally, to the molecular chirality has remained elusive. Here, a method to determine the atomic contributions to the optical rotation angle is reported. This approach links chemical structure with optical rotation angle and provides a quantitative measure of molecular asymmetry propagation from a center, axis, or plane of chirality.
Thiol additions to the highly functionalized core structures of aranorosin- and manumycin-type antibiotics reveal the general reactivity patterns of epoxyketone natural products. Rapid hemiacetal and hydrate formations decrease the reactivity of the epoxyketone moiety in aqueous media toward the cellular scavenger glutathione, and secondary 1,2-shift, elimination, aromatization and intramolecular aldol reactions provide novel reaction pathways. In a hydrophobic environment, the thiol-capture function of the ketone moiety facilitates electrophilic attack.
We previously reported the generation of a library of hydrophobic oxazole-based small molecules designed as inhibitors of phosphatases involved in cellular signaling and cell cycle control. One member of the targeted array library, 4-(benzyl-(2-[(2, 5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylami no butyric acid (SC-alphaalphadelta9), inhibited cell growth in the G0/G1 phase of the cell cycle. To investigate potential mechanisms for SC-alphaalphadelta9 antiproliferative activity, we have used mouse embryonic fibroblasts transformed with simian virus 40 large T antigen mouse embryonic fibroblasts as a model system for a malignant phenotype that depends on overexpression of cell cycle regulators and autocrine stimulation by insulin-like growth factor-1. Structure-activity relationship studies with SC-alphaalphadelta9 and four library congeners demonstrated that antiproliferative activity was not a result of overall hydrophobicity. Rather, SC-alphaalphadelta9 decreased insulin-like growth factor-1 receptor tyrosine phosphorylation, receptor expression, mitogen-activated protein kinase activation and levels of the cyclin-dependent kinase Cdc2. Less toxic congeners only partially affected receptor expression, receptor tyrosine phosphorylation and Cdc2 levels. Thus SC-alphaalphadelta9, which is structurally distinct from other known small molecules that decrease intracellular Cdc2 levels, has profound effects on intracellular signaling. Furthermore, SC-alphaalphadelta9, but not vanadate or okadaic acid, selectively inhibited the growth of simian virus 40 large T antigen mouse embryonic fibroblasts compared to the parental cells. These results suggest that overexpression of Cdc2 and increased dependence on insulin-like growth factor-1 autocrine stimulation are responsible for the increased sensitivity of simian virus 40 large T antigen mouse embryonic fibroblasts to SC-alphaalphadelta9. The SC-alphaalphadelta9 pharmacophore could be a useful platform for the development of novel antisignaling agents.
Tyrosine phosphatases (PTPases) dephosphorylate phosphotyrosines while dual-specificity phosphatases (DSPases) dephosphorylate contiguous and semicontiguous phosphothreonine and phosphotyrosine on cyclin dependent kinases and mitogen-activated protein kinases. Consequently, PTPases and DSPases have a central role controlling signal transduction and cell cycle progression. Currently, there are few readily available potent inhibitors of PTPases or DSPases other than vanadate. Using a pharmacophore modeled on natural product inhibitors of phosphothreonine phosphatases, we generated a refined library of novel, phosphate-free, small-molecule compounds synthesized by a parallel, solid-phase combinatorial-based approach. Among the initial 18 members of this targeted diversity library, we identified several inhibitors of DSPases: Cdc25A, -B, and -C and the PTPase PTP1B. These compounds at 100 microM did not significantly inhibit the protein serine/threonine phosphatases PP1 and PP2A. Kinetic studies with two members of this library indicated competitive inhibition for Cdc25 DSPases and noncompetitive inhibition for PTP1B. Compound AC-alphaalpha69 had a Ki of approximately 10 microM for recombinant human Cdc25A, -B, and -C, and a Ki of 0.85 microM for the PTP1B. The marked differences in Cdc25 inhibition as compared to PTP1B inhibition seen with relatively modest chemical modifications in the modular side chains demonstrate the structurally demanding nature of the DSPase catalytic site distinct from the PTPase catalytic site. These results represent the first fundamental advance toward a readily modifiable pharmacophore for synthetic PTPase and DSPase inhibitors and illustrate the significant potential of a combinatorial-based strategy that supplements the rational design of a core structure by a randomized variation of peripheral substituents.
Recovery and purification difficulties can limit the yield and utility of otherwise successful organic synthesis strategies. A "fluorous synthesis" approach is outlined in which organic molecules are rendered soluble in fluorocarbon solvents by attachment of a suitable fluorocarbon group. Fluorocarbon solvents are usually immiscible in organic solutions, and fluorous molecules partition out of an organic phase and into a fluorous phase in a standard liquid-liquid extraction. Simple yet substantive separations of organic reaction mixtures are achieved without resorting to chromatography. Because fluorous synthesis combines in many respects the favorable purification features of solid-phase synthesis with the favorable reaction, identification, and analysis features of traditional organic synthesis, it should prove valuable in the automated synthesis of libraries of individual pure organic compounds.
When plane-polarized light impinges on a solution of optically active molecules, the polarization of the light that emerges is rotated. This simple phenomenon arises from the interaction of light with matter and is well understood, in principle, van't Hoff's rule of optical superposition correlates the molar rotation with the individual contributions to optical activity of isolated centers of asymmetry. This straightforward empirical additivity rule is rarely used for structure elucidation nowadays because of its limitations in the assessment of conformationally restricted or interacting chiral centers. However, additivity can be used successfully to assign the configuration of complex natural products such as hennoxazole A if appropriate synthetic partial structures are available. Therefore, van't Hoff's principle is a powerful stereochemical complement to natural products' total synthesis. The quest for reliable quantitative methods to calculate the angle of rotation a priori has been underway for a long time. Both classical and quantum methods for calculating molar rotation have been developed. Of particular practical importance for determining the absolute structure of molecules by calculation is the manner in which interactions between multiple chiral centers in a single molecule are included, leading to additive or non-additive optical rotation angles. This problem is addressed here using semi-empirical electronic structure models and the Rosenfeld equation.
In eukaryotes, phosphorylation of serine, threonine, and tyrosine residues on proteins is a fundamental posttranslational regulatory process for such functions as signal transduction, gene transcription, RNA splicing, cellular adhesion, apoptosis, and cell cycle control. Based on functional groups present in natural product serine/threonine protein phosphatase (PSTPase) inhibitors, we have designed pharmacophore model 1 and demonstrated the feasibility of a combinatorial chemistry approach for the preparation of functional analogues of 1. Preliminary biological testing of 18 structural variants of 1 has identified two compounds with growth inhibitory activity against cultured human breast cancer cells. In vitro inhibition of the PSTPase PP2A was demonstrated with compound 1d. Using flow cytometry we observed that compound f1 caused prominent inhibition in the G1 phase of the cell cycle. Thus, the combinatorial modifications of the minimal pharmacophore 1 can generate biologically interesting antiproliterative agents.
N4-Dipeptidyl slow-release forms of the anticancer drug ara-C were prepared by acylation of the lithiated nucleotide with 4,4-dialkyloxazolinones. An azapeptide prodrug of ara-C was obtained by condensation of an amino acid hydrazide with an activated nucleotide urea. The use of unnatural amino acid residues at N4 prevented nonspecific proteolytic cleavage in biological medium. Ara-C prodrugs 10, 15, 17, and 19 released active drug with half-lives from a few minutes to several days, respectively. Activation via intramolecular N4-deacylation did not require enzymatic intervention but was strictly dependent on the structure of the peptide chain. The prodrugs 10, 15, and 17 produced similar growth inhibition as ara-C in cultured murine leukemia cells while the azapeptide prodrug 19 was less potent reflecting the slow release of active drug with this compound. All four prodrugs retained the ability to induce apoptosis in human HL-60 leukemia cells with kinetics dictated by the rate of intramolecular N4-deacylation. This the first demonstration for the control of apoptotic cell death by the modulation of drug release from prodrugs.