Oxindole-based inhibitors of cyclin-dependent kinase 2 (CDK2): design, synthesis, enzymatic activities, and X-ray crystallographic analysis.

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.

Prevention of chemotherapy-induced alopecia in rats by CDK inhibitors.

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.

Antitumor efficacy, pharmacokinetics, and biodistribution of NX 211: a low-clearance liposomal formulation of lurtotecan.

Lurtotecan is a clinically active water-soluble camptothecin analogue that has been formulated into a low-clearance unilamellar liposome, NX 211. Comparative studies between free drug and NX 211 have been performed assessing pharmacokinetics in nude mice, tissue distribution in tumor-bearing mice, and antitumor efficacy in xenografts. Compared with lurtotecan, NX 211 demonstrated a significant increase in plasma residence time and a subsequent 1500-fold increase in the plasma area under the drug concentration curve. The volume of distribution was also greatly restricted, suggesting altered tissue distribution. Evaluation of tissues 24 h after administration of either [14C]NX 211 or [14C]lurtotecan to ES-2 tumor-bearing mice demonstrated a 40-fold increase in radiolabeled compound in the tumors of NX 211-treated mice compared with mice treated with lurtotecan. In single-dose efficacy studies, NX 211 produced a consistent 3-fold or greater increase in therapeutic index compared with lurtotecan in both the KB and ES-2 xenograft models. When compared at equitoxic levels in repeat-dose efficacy studies, NX 211 generated durable cures lasting >60 days and a 2-8-fold increase in log10 cell kill, compared with lurtotecan and topotecan, respectively. Together, these data demonstrate that NX 211 has significant therapeutic advantage over lurtotecan and that the improved antitumor activity is consistent with increased exposure and enhanced drug delivery to tumor sites.

Novel 1,3-disubstituted-5,10-dihydro-5,10-dioxo-1H-benzo[g] isochromene-3 carboxamides as potent antitumor agents.

Novel antitumor 5,10-dihydro-5,10-dioxo-1H- benzo[g]isochromene-3-carboxamides were discovered.

Water soluble inhibitors of topoisomerase I: quaternary salt derivatives of camptothecin.

Eleven water soluble 7-substituted quaternary ammonium salt derivatives of 10,11-(methylenedioxy)- and 10,11-(ethylenedioxy)-(20S)-camptothecin were synthesized via the Friedlander reaction followed by nucleophilic displacement with an aromatic amine. All of these compounds were more potent than camptothecin in the in vitro cleavable complex assay. These inherently charged camptothecin derivatives were cytotoxic against three different human tumor cell lines (SKOV3, an ovarian adenocarcinoma; SKVLB a multidrug resistant ovarian adenocarcinoma; and HT-29, a colon carcinoma). A selected group of five compounds was evaluated in the nude mouse HT-29 xenograft model. Two of these quaternary salts (17 and 18) were more efficacious than Topotecan in delaying tumor growth. In an extended in vivo model, 18 demonstrated tumor regression.

Synthesis, topoisomerase I inhibitory activity, and in vivo evaluation of 11-azacamptothecin analogs.

A series of analogs based on a novel template, 11-aza-(20S)-camptothecin, were obtained from total synthesis and tested as potential anticancer drugs in the topoisomerase I enzyme cleavable complex assay. The parent compound 11-aza-(20S)-camptothecin (8) was derived from a Friedlander condensation between the known aminopyridine derivative 3-(3-amino-4-picolylidene)-p-toluidine and optically active tricyclic ketone 7. Compound 8 had activity approximately twice that of (20S)-camptothecin in the calf thymus topoisomerase I cleavable complex assay. Compounds were prepared wherein the 11-aza nitrogen atom was quaternized as either the corresponding N-oxide or methyl iodide. Compounds with quaternized N-11 showed improved water solubility and were equipotent to the clinically investigated camptothecin analog topotecan in the cleavable complex assay. These compounds were evaluated in vivo in nude mice bearing HT-29 human colon carcinoma xenografts. The analog 11-aza-(20S)-camptothecin 11-N-oxide was found to significantly retard tumor growth when compared to untreated controls. Finally, 7,10-disubstituted 11-azacamptothecin analogs were synthesized using Pd(0) coupling reactions of 10-bromo-7-alkyl-11-aza-(20S)-camptothecins 19 and 20, which in turn were available from a Friedlander condensation of the novel bromopyridine derivatives 17a and 17b with 7. Among the 10-substituted series, a number of analogs displayed extremely high in vitro potency against topoisomerase I and improved aqueous solubility. A significant number of the compounds were found to be active in whole cell cytotoxicity assays and several were evaluated in nude mice bearing the HT-29 tumor xenografts. The most effective of these proved to be (S)-11-aza-7-ethyl-10-(aminohydroximinomethyl)camptothecin trifluoracetic acid salt (27), a potent topoisomerase I inhibitor which demonstrated excellent efficacy in both short term and in extended in vivo assays. A comparison between in vitro enzyme data and in vivo data from nude mouse studies in other compounds in this series revealed a poor overall correlation between topoisomerase inhibition in vitro and antitumor efficacy in vivo.

Synthesis and antitumor activity of novel water soluble derivatives of camptothecin as specific inhibitors of topoisomerase I.

The synthesis and antitumor activities of the novel water soluble camptothecin derivatives 7-[(4-methylpiperazino)methyl]-10,11-(methylenedioxy)-(20S)-campto thecin trifluoroacetate (6) and 7-[(4-methylpiperazino)methyl]-10,11-(ethylenedioxy)-(20S)-camptot hecin trifluoroacetate (7) are described. The solubilities of compounds 6 and 7 were measured to be 4.5 and 5.8 mg/mL, respectively, in pH 5 acetate buffer in contrast to < 0.003 mg/mL for camptothecin in the same buffer. In the purified topoisomerase I cleavable complex enzyme assay, compounds 6 and 7 demonstrated potent inhibition of topoisomerase I with IC50's of 300 and 416 nM, respectively, in comparison to 679 nM for camptothecin and 1028 nM for topotecan. In human tumor cell cytotoxicity assays, compounds 6 and 7 demonstrated potent antitumor activity against ovarian (SKOV3), ovarian with upregulated MDRp-glycoprotein (SKVLB), melanoma (LOX), breast (T47D), and colon (HT29) with IC50's ranging from 0.5 to 102 nM. Compounds 6 and 7 induced tumor regressions in the HT29 human colon tumor xenograft model and demonstrated similar rank order of potency compared to in vitro assay results.

In vivo antitumor activity of two new seven-substituted water-soluble camptothecin analogues.

The development of camptothecin-like compounds as inhibitors of topoisomerase I for the treatment of resistant tumors has generated clinical excitement in this new class of drugs. We have developed two novel water-soluble camptothecin analogues which are specific inhibitors of topoisomerase I and are potent cytotoxins with significant antitumor activity. We added water-solubilizing groups off position 7 in the B ring of either 10,11-ethylenedioxy- or 10,11-methylenedioxy-20(S)-camptothecin. These water-soluble camptothecin analogues were demonstrated to be nanamolar inhibitors of the topoisomerase I enzyme in the cleavable complex assay. The compounds, GI147211 [7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camp tot hecin], and GI149893 [7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)-cam pto thecin], were compared to topotecan, a known water-soluble inhibitor of topoisomerase I. Both GI compounds were found to be slightly more potent than topotecan as inhibitors of topoisomerase I in the cleavable complex assay and were 1.5-2 times more soluble. Tumor cell cytotoxicity assays using 5 separate cell lines demonstrated that both GI compounds were 5-10 times more potent than topotecan, although by comparison all three topoisomerase I inhibitors were unaffected by the multidrug resistance P-glycoprotein. The antitumor activity of all three topoisomerase I inhibitors was compared concomitantly in two human colon xenograft models. In both models, GI147211 and GI149893 were able to induce regression of established HT-29 and SW-48 colon tumors by as much as 60%. The antitumor activity of both compounds were also demonstrated in the MX-1 and PC-3 xenografts. Microscopic examination of selected tissues indicated that drug-induced toxicity was primarily limited to the gastrointestinal tract and was comparable among the three compounds. Further clinical development of this class of compounds is ongoing.