Bcl-2 and related proteins are key regulators of apoptosis or programmed cell death implicated in human disease including cancer. We recently showed that cell-permeable Bcl-2 binding peptides could induce apoptosis of human myeloid leukemia in vitro and suppress its growth in severe combined immunodeficient mice. Here we report the discovery of HA14-1, a small molecule (molecular weight ‫؍‬ 409) and nonpeptidic ligand of a Bcl-2 surface pocket, by using a computer screening strategy based on the predicted structure of Bcl-2 protein. In vitro binding studies demonstrated the interaction of HA14-1 with this Bcl-2 surface pocket that is essential for Bcl-2 biological function. HA14-1 effectively induced apoptosis of human acute myeloid leukemia (HL-60) cells overexpressing Bcl-2 protein that was associated with the decrease in mitochondrial membrane potential and activation of caspase-9 followed by caspase-3. Cytokine response modifier A, a potent inhibitor of Fas-mediated apoptosis, did not block apoptosis induced by HA14-1. Whereas HA14-1 strongly induced the death of NIH 3T3 (Apaf-1 ؉͞؉ ) cells, it had little apoptotic effect on Apaf-1-deficient (Apaf-1 ؊͞؊ ) mouse embryonic fibroblast cells. These data are consistent with a mechanism by which HA14-1 induces the activation of Apaf-1 and caspases, possibly by binding to Bcl-2 protein and inhibiting its function. The discovery of this cell-permeable molecule provides a chemical probe to study Bcl-2-regulated apoptotic pathways in vivo and could lead to the development of new therapeutic agents.

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shown by GLC analysis and 13C NMR to contain 5a (lo%), unknown compounds (12%), and 3a (78%). The data (GLC and '3c NMR) are identical with those from the products from reaction of 1 with methanol a t room temperature. Acknowledgment. We acknowledge grants from the Research Committee of the New Zealand Universities J. Org. Chem. 1990,55, 4144-4153 Grants Committee and helpful discussions with Professor M. A. Battiste.Supplementary Material Available: 13C and 'H NMR spectra for compounds 1,3a-d, 5a,b, 6a,b, 12,14a-c, 16a,b, and 18a,b (26 pages). Ordering information is given on any current masthead page.The carbenoid intermediate derived by treating ethyl 2-diazo-4-phthalimidobutyrate with rhodium(I1) octanoate undergoes transannular cyclization onto the adjacent imido carbonyl group. The resulting cyclic carbonyl ylide dipole was trapped with several dipolarophiles. In an attempt to prepare related substrates for cyclization studies, the reaction of ethyl lithiodiazoacetate with various aldehydes and ketones was studied. Treatment of the a-diazo-0-hydroxy ester derived from acetone or cyclopentanone with rhodium(I1) octanoate gave rise to a 0-keto ester. The exclusive phenyl shift encountered with acetophenone is in keeping with migration to an electron-deficient center. The reaction works well with acrolein, leading to high yields of 3-oxo-4-pentenoate. The 1,2-hydrogen shift pathway was found to proceed much faster than intramolecular cyclopropanation. Dehydration of the a-diazo-@-hydroxy esters generates vinyl diazo esters, which readily cyclize to lH-pyrazoles on thermolysis.

Mutations in the TP53 tumor suppressor gene have been studied in different types of brain tumors. Little is known about this genetic event in human meningioma, a mostly benign tumor. To investigate the frequency of TP53 gene mutations in human tumors derived from meningeal tissues, paraffin-embedded tissues from 30 cases (including 2 malignant and 4 atypical meningiomas, as well as 2 hemangioblastomas and 3 hemangiopericytomas) were screened by immunohistochemistry. Polymerase chain reaction/single strand conformational polymorphism (PCR/SSCP) and direct DNA sequencing were thereafter performed in selected cases. Nuclear p53 staining was not seen in any of the 19 benign meningiomas tested, while atypical meningiomas, hemangioblastomas, and hemangiopericytomas displayed nuclear staining in a subpopulation of tumor cells in 4 out of 5, 2 out of 2, and 3 out of 3 cases, respectively. One malignant meningioma showed an intense nuclear staining and a band shift in SSCP. In this case, we identified a mutation in the TP53 gene at codon 161 changing GCC to ACC and resulting in an alteration of alanine to threonine in this position. Our results indicate that TP53 gene mutation may be considered as a marker for malignant transformation in meningioma. p53 immunoreactivity, even in the absence of detectable gene mutation, is also associated with atypia and does not appear in regular benign meningiomas.