Paullones, a Series of Cyclin-Dependent Kinase Inhibitors: Synthesis, Evaluation of CDK1/Cyclin B Inhibition, and in Vitro Antitumor Activity Christiane Schultz, ‡ Andreas Link, ‡ Maryse Leost, § Daniel W. Zaharevitz, † Rick Gussio, † Edward A. Sausville, † Laurent Meijer, § and Conrad Kunick* ,‡ Institut fu ¨ r Pharmazie, Abteilung fu ¨ r Pharmazeutische Chemie, Universita ¨ t Hamburg, Bundesstrasse 45, D-20146 Hamburg, Germany, Centre National de la Recherche Scientifique, Station Biologique, BP 74, F-29682 Roscoff, France, and Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, Maryland 20852 Received February 2, 1999 The paullones represent a novel class of small molecule cyclin-dependent kinase (CDK) inhibitors. To investigate structure-activity relationships and to develop paullones with antitumor activity, derivatives of the lead structure kenpaullone (9-bromo-7,12-dihydroindolo- [3,2-d][1]benzazepin-6(5H)-one, 4a) were synthesized. Paullones with different substituents in the 2-, 3-, 4-, 9-, and 11-positions were prepared by a Fischer indole reaction starting from 1H-[1]benzazepine-2,5(3H,4H)-diones 5. Selective substitutions at either the lactam or the indole nitrogen atom were accomplished by treating kenpaullone with alkyl halides in the presence of sodium hydride/THF or potassium hydroxide/acetone, respectively. S-Methylation of the kenpaullone-derived thiolactam 18 yielded the methylthioimidate 19, which gave the hydroxy- amidine 20 upon reaction with hydroxylamine. The new paullones were tested both in a CDK1/ cyclin B inhibition assay and in the in vitro antitumor cell line-screening program of the National Cancer Institute (NCI). With respect to the CDK1/cyclin B inhibition, electron- withdrawing substituents in the 9-position as well as a 2,3-dimethoxy substitution on the paullone basic scaffold turned out to be favorable. A 9-trifluoromethyl substituent was found to be equivalent to the 9-bromo substituent of kenpaullone. Replacement of the 9-bromo substituent of kenpaullone by a 9-cyano or 9-nitro group produced a substantial increase in enzyme-inhibiting potency. Substitutions in other positions or the replacement of the lactam moiety led to decreased CDK1 inhibition. Noteworthy in vitro antitumor activities (GI 50 values between 1 and 10 µM) were found with the 9-bromo-2,3-dimethoxy-7,12-dihydroindolo[3,2-d]- [1]benzazepin-6(5H)-one (4t), its 9-trifluoromethyl analogue 4u, the 12-Boc-substituted paullone 15, and the methylthioimidate 19, respectively. The 9-nitro-7,12-dihydroindolo[3,2-d][1]- benzazepin-6(5H)-one (4j, named alsterpaullone) showed a high CDK1/cyclin B inhibitory activity (IC 50 ) 0.035 µM) and exceeded the in vitro antitumor potency of the other paullones by 1 order of magnitude (log GI 50 mean graph midpoint )-6.4 M). Introduction The cell division cycle is driven and regulated by a variety of complex processes. Among the manifold molecular entities that are involved in the surveillance of the cell cycle, the cyclin-dependent kinases (CDKs) play a central role. The CDKs are a group of serine threonine kinases, which control the transmission be- tween successive stages of the cell cycle. 1 The activity of the CDKs is regulated by multiple mechanisms, including binding to cyclins, a diverse class of positive regulatory CDK-binding proteins. The oscillating con- centration of the cyclins during the cell cycle is the basis for the stage-dependent activity of the CDKs. Binding to CDK inhibitory proteins (CKIs) results in deactiva- tion of CDKs. In various human tumors, deregulations of CDK-related mechanisms have been found, e.g., overexpression of cyclins or deletion of genes encoding for CKIs. 2-5 Considering these observations, CDKs are attractive targets for the development of antitumor drugs. 6-9 The number of chemical agents that act selectively as CDK inhibitors is limited; among them are a lactone (butyrolactone I (1)), flavonoids (e.g., flavopiridol (2)), and several purine derivatives (Chart 1). Butyrolactone I(1) has shown antiproliferative activity for colon and pancreatic carcinoma cell lines. 10,11 Flavopiridol (2) is the first CDK inhibitor that has entered clinical trials as an anticancer agent. 12,13 The class of purine-related derivatives comprises the majority of known CDK inhibitors, e.g., olomoucine, 14 roscovitine (3), 15,16 and the purvalanols. 17 X-ray structure determination of crystal- lized inhibitor-enzyme complexes revealed that the purine-derived compounds as well as flavopiridol bind to the ATP-binding pocket of the CDKs. 12,17-20 When flavopiridol was used in a COMPARE search 21 performed at the National Cancer Institute (NCI) in a database of compounds tested in the NCI in vitro cancer cell line screening, the 9-bromo-7,12-dihydroindolo[3,2- d][1]benzazepin-6(5H)-one (4a, kenpaullone) 22 was iden- tified as a potential CDK inhibitor. 23 Inhibition experi- ments revealed that 4a indeed is a potent inhibitor of CDKs with selectivity for CDK1, CDK2, and CDK5. It was shown, that kenpaullone acts as an ATP competi- ‡ Universita ¨ t Hamburg. § Centre National de la Recherche Scientifique. † National Cancer Institute. 2909 J. Med. Chem. 1999, 42, 2909-2919 10.1021/jm9900570 CCC: $18.00 © 1999 American Chemical Society Published on Web 06/30/1999