Two closely related classes of oxindole-based compounds, 1H-indole-2,3-dione 3-phenylhydrazones and 3-(anilinomethylene)-1,3-dihydro-2H-indol-2-ones, were shown to potently inhibit cyclin-dependent kinase 2 (CDK2). The initial lead compound was prepared as a homologue of the 3-benzylidene-1,3-dihydro-2H-indol-2-one class of kinase inhibitor. Crystallographic analysis of the lead compound bound to CDK2 provided the basis for analogue design. A semiautomated method of ligand docking was used to select compounds for synthesis, and a number of compounds with low nanomolar inhibitory activity versus CDK2 were identified. Enzyme binding determinants for several analogues were evaluated by X-ray crystallography. Compounds in this series inhibited CDK2 with a potency approximately 10-fold greater than that for CDK1. Members of this class of inhibitor cause an arrest of the cell cycle and have shown potential utility in the prevention of chemotherapy-induced alopecia.

The antiproliferative effects of interferon a (IFN-a) and interferon y (IFN-y) were found to be celldependent. Among the human cell lines examined, IFN-y had a greater antiproliferative effect against cell lines that exhibited induction of indoleamine 2,3-dioxygenase, such as the KB oral carcinoma or WiDr colon adenocarcinoma, than against those that lacked the enzyme activity, such as the SW480 colon adenocarcinoma or NCI-H128 small-cell lung carcinoma. MATERIALS AND METHODSCell Culture. Human cell lines HL-60 (promyelocytic leukemia), KG-1 (myelogenous leukemia), CCRF-SB (B-cell leukemia), CCRF-CEM (T-cell, leukemia), MOLT-4 (T-cell leukemia), THP-1 (monocytic leukemia), U-937 (histiocytic lymphoma), NCI-H69 and NCI-H128 (small-cell lung carcinomas), A-427 (lung carcinoma), MRC-5 (normal fetal lung), HEp-2 (larynx carcinoma), KB (oral carcinoma), MCF7 (breast adenocarcinoma), SW480 (colon adenocarcinoma), WiDr (colon adenocarcinoma), and I407 (normal embryonic intestine) were obtained from the American Type Culture Collection. The cells (0.3-1 x 106) were grown in 60-mm plastic culture plates (Costar, Cambridge, MA) containing 6 ml of culture medium consisting of 25 mM Hepes-buffered RPMI 1640 (GIBCO) with 10% heat-inactivated fetal bovine serum, penicillin (50 units/ml), and streptomycin (0.05 mg/ ml; Hazleton Research Products, Lenexa, KS) in the presence of IFN-a

Most traditional cytotoxic anticancer agents ablate the rapidly dividing epithelium of the hair follicle and induce alopecia (hair loss). Inhibition of cyclin-dependent kinase 2 (CDK2), a positive regulator of eukaryotic cell cycle progression, may represent a therapeutic strategy for prevention of chemotherapy-induced alopecia (CIA) by arresting the cell cycle and reducing the sensitivity of the epithelium to many cell cycle-active antitumor agents. Potent small-molecule inhibitors of CDK2 were developed using structure-based methods. Topical application of these compounds in a neonatal rat model of CIA reduced hair loss at the site of application in 33 to 50% of the animals. Thus, inhibition of CDK2 represents a potentially useful approach for the prevention of CIA in cancer patients.

Communicated by George H. Hitchings, December 13, 1982 ABSTRACT Mammalian cells and tissues were found to have two pathways for the biosynthesis of tetrahydrobiopterin (BH4): (i) the conversion of GTP to BH4 by a methotrexate-insensitive de novo pathway, and (ii) the conversion of sepiapterin to BH4 by a pterin salvage pathway dependent on dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) activity. In a Chinese hamster ovary cell mutant lacking dihydrofolate reductase (DUKX-BII), endogenous formation of BH4 proceeds normally but, unlike the parent cells, these cells or extracts ofthem do not convert sepiapterin or 7,8-dihydrobiopterin to BH4. KB cells, which do not contain detectable levels of GTP cyclohydrolase or BH4 but do contain dihydrofolate reductase, readily convert sepiapterin to BH4 and this conversion is completely prevented by methotrexate. In supernatant fractions of bovine adrenal medulla, the conversion of sepiapterin to BH4 is completely inhibited by methotrexate. Similarly, this conversion in rat brain in vivo is methotrexate-sensitive. Sepiapterin and 7,8-dihydrobiopterin apparently do not enter the de novo pathway of BH4 biosynthesis and may be derived from labile intermediates which have not yet been characterized.