Radiosensitization of p53 mutant cells by PD0166285, a novel G(2) checkpoint abrogator.

The lack of functional p53 in many cancer cells offers a therapeutic target for treatment. Cells lacking p53 would not be anticipated to demonstrate a G(1) checkpoint and would depend on the G(2) checkpoint to permit DNA repair prior to undergoing mitosis. We hypothesized that the G(2) checkpoint abrogator could preferentially kill p53-inactive cancer cells by removing the only checkpoint that protects these cells from premature mitosis in response to DNA damage. Because Wee1 kinase is crucial in maintaining G(2) arrest through its inhibitory phosphorylation of Cdc2, we developed a high-throughput mass screening assay and used it to screen chemical library for Wee1 inhibitors. A pyridopyrimidine class of molecule, PD0166285 was identified that inhibited Wee1 at a nanomolar concentration. At the cellular level, 0.5 microM PD0166285 dramatically inhibits irradiation-induced Cdc2 phosphorylation at the Tyr-15 and Thr-14 in seven of seven cancer cell lines tested. PD0166285 abrogates irradiation-induced G(2) arrest as shown by both biochemical markers and fluorescence-activated cell sorter analysis and significantly increases mitotic cell populations. Biologically, PD0166285 acts as a radiosensitizer to sensitize cells to radiation-induced cell death with a sensitivity enhancement ratio of 1.23 as shown by standard clonogenic assay. This radiosensitizing activity is p53 dependent with a higher efficacy in p53-inactive cells. Thus, G(2) checkpoint abrogators represent a novel class of anticancer drugs that enhance cell killing of conventional cancer therapy through the induction of premature mitosis.

Differential utilization and localization of ErbB receptor tyrosine kinases in skin compared to normal and malignant keratinocytes.

Induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA in mouse skin organ culture was blocked by two pan-ErbB receptor tyrosine kinase (RTK) inhibitors but not by genetic ablation of ErbB1, suggesting involvement of multiple ErbB species in skin physiology. Human skin, cultured normal keratinocytes, and A431 skin carcinoma cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4. Skin and A431 cells expressed more ErbB3 than did keratinocytes. Despite strong expression of ErbB2 and ErbB3, heregulin was inactive in stimulating tyrosine phosphorylation in A431 cells. In contrast, it was highly active in MDA-MB-453 breast carcinoma cells. ErbB2 displayed punctate cytoplasmic staining in A431 and keratinocytes, compared to strong cell surface staining in MDA-MB-453. In skin, ErbB2 was cytoplasmic in basal keratinocytes, assuming a cell surface pattern in the upper suprabasal layers. In contrast, ErbB1 retained a cell surface distribution in all epidermal layers. Keratinocyte proliferation in culture was found to be ErbB1-RTK-dependent, using a selective inhibitor. These results suggest that in skin keratinocytes, ErbB2 transduces ligand-dependent differentiation signals, whereas ErbB1 transduces ligand-dependent proliferation/survival signals. Intracellular sequestration of ErbB2 may contribute to the malignant phenotype of A431 cells, by allowing them to respond to ErbB1-dependent growth/survival signals, while evading ErbB2-dependent differentiation signals.

Cell cycle and biochemical effects of PD 0183812. A potent inhibitor of the cyclin D-dependent kinases CDK4 and CDK6.

Progression through the G1 phase of the cell cycle requires phosphorylation of the retinoblastoma gene product (pRb) by the cyclin D-dependent kinases CDK4 and CDK6, whose activity can specifically be blocked by the CDK inhibitor p16(INK4A). Misregulation of the pRb/cyclin D/p16(INK4A) pathway is one of the most common events in human cancer and has lead to the suggestion that inhibition of cyclin D-dependent kinase activity may have therapeutic value as an anticancer treatment. Through screening of a chemical library, we initially identified the [2,3-d]pyridopyrimidines as inhibitors of CDK4. Chemical modification resulted in the identification of PD 0183812 as a potent and highly selective inhibitor of both CDK4 and CDK6 kinase activity, which is competitive with ATP. Flow cytometry experiments showed that of the cell lines tested, only those expressing pRb demonstrated a G1 arrest when treated with PD 0183812. This arrest correlated in terms of incubation time and potency with a loss of pRb phosphorylation and a block in proliferation, which was reversible. These results suggest a potential use of this chemical class of compounds as therapeutic agents in the treatment of tumors with functional pRb, possessing cell cycle aberrations in other members of the pRb/cyclin D/p16(INK4A) pathway.

3-(3,5-Dimethoxyphenyl)-1,6-naphthyridine-2,7-diamines and related 2-urea derivatives are potent and selective inhibitors of the FGF receptor-1 tyrosine kinase.

A series of 3-aryl-1,6-naphthyridine-2,7-diamines and related 2-ureas were prepared and evaluated as inhibitors of the FGF receptor-1 tyrosine kinase. Condensation of 4,6-diaminonicotinaldehyde and substituted phenylacetonitriles gave intermediate naphthyridine-2,7-diamines, and direct reaction of the monoanion of these (NaH/DMF) with alkyl or aryl isocyanates selectively gave the 2-ureas in varying yields (23-93%). For the preparation of more soluble 7-alkylamino-2-ureas, a number of protecting groups for the 2-amine were evaluated (phthaloyl, 4-methoxybenzyl) following selective blocking of the 7-amine (trityl), but these were not superior to the (required) 2-tert-Bu-urea group itself. Direct alkylation of the anion of the (unprotected) 7-amino group with excess 4-(3-chloropropyl)morpholine in DMF gave low (10%) yields of the desired product, but alkylation of the 7-acetamido anion, followed by mild alkaline hydrolysis, raised this to 64%. 3-Phenyl analogues were nonspecific inhibitors of isolated c-Src, FGFR, and PDGFR tyrosine kinases, whereas 3-(2,6-dichlorophenyl) analogues were most effective against c-Src and FGFR, and 3-(3,5-dimethoxyphenyl) derivatives showed high selectivity for FGFR alone. A water-soluble (7-morpholinylpropylamino) analogue retained high FGFR potency (IC(50) 31 nM) and selectivity. Pairwise comparison of the 1, 6-naphthyridines and the corresponding known pyrido[2,3-d]pyrimidine analogues showed little differences in potency or patterns of selectivity, suggesting that the 1-aza atom of the latter is not important for activity. A 7-acetamide derivative inhibited the growth of FGFR-expressing tumor cell lines and was particularly potent against HUVECs (IC(50) 4 nM). This compound was also a very potent inhibitor of HUVEC microcapillary formation (IC(50) 0.01 nM) and Matrigel invasion (IC(50) 7 nM) and showed significant in vivo antitumor effects in a highly vascularized mammary adenocarcinoma 16/c model at nontoxic doses. The compounds are worthy of further evaluation as antiangiogenesis agents.

Anticancer efficacy of the irreversible EGFr tyrosine kinase inhibitor PD 0169414 against human tumor xenografts.

PURPOSE: The involvement of the EGF receptor (EGFr) family of receptors in cancers suggests that a selective inhibitor of the tyrosine kinase activity of the EGFr family could have a therapeutic effect. PD 0169414, an anilinoquinazoline, is a potent irreversible inhibitor of the EGFr family tyrosine kinase activity with IC(50) values of 0.42 nM against the isolated EGF receptor, and 4.7 nM and 22 nM against EGF- and heregulin-mediated receptor phosphorylation in A431 and MDA-MB-453 cells, respectively. METHODS AND RESULTS: Oral administration of 260 mg/kg per day PD 0169414 for 15 days to animals bearing advanced-stage A431 epidermoid carcinoma produced a 28.2-day delay in tumor growth and resulted in three complete and three partial tumor regressions in six animals. Toxicity at this dose level was limited to <6% loss of initial body weight. Doses of 160 and 100 mg/kg per day produced tumor growth delays of 29.5 and 20.9 days and two and one complete regressions in six animals, respectively. Subcutaneous, intraperitoneal, and oral routes of administration have also shown in vivo antitumor activity of PD 0169414 in a panel of human tumor xenografts. Responsive tumor lines include A431 (human epidermoid carcinoma), H125 (NSCL carcinoma), MCF-7 and UISO-BCA1 (human breast carcinoma), and SK-OV-03 (human ovarian carcinoma). The therapeutic effect ranged from delayed tumor growth (6.4 days delayed tumor growth for 14 days of treatment) to tumor regressions (32.2 days delayed tumor growth and five partial regressions in six animals) in these model systems. CONCLUSION: PD 0169414 is a specific, irreversible inhibitor of EGFr family tyrosine kinases with significant in vivo activity against a variety of relevant human tumor xenografts.

Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo.

The mitogen-activated protein kinase pathway is thought to be essential in cellular growth and differentiation. Here we report the discovery of a highly potent and selective inhibitor of the upstream kinase MEK that is orally active. Tumor growth was inhibited as much as 80% in mice with colon carcinomas of both mouse and human origin after treatment with this inhibitor. Efficacy was achieved with a wide range of doses with no signs of toxicity, and correlated with a reduction in the levels of activated mitogen-activated protein kinase in excised tumors. These data indicate that MEK inhibitors represent a promising, noncytotoxic approach to the clinical management of colon cancer.

Growth inhibition of nasopharyngeal carcinoma cells by EGF receptor tyrosine kinase inhibitors.

Nasopharyngeal carcinoma (NPC) is a malignancy of epithelial origin occurring with a high incidence in southern China and southeast Asia. Radiotherapy is the main treatment modality for NPC. No effective chemotherapy is available. Since prevention of EGF/EGFR binding by an EGFR specific monoclonal antibody suppressed the growth of NPC xenografts, we examined potential anti-NPC activity by a group of specific inhibitors of the EGFR family of tyrosine kinases. We found that HONE-T1 NPC cells expressed high levels of EGFR tyrosine kinase activity upon stimulation by EGF. The receptor tyrosine kinase activity was specifically inhibited by either reversible (PD158780) or irreversible (PD168393) inhibitors specific for EGFR family tyrosine kinases. This inhibition led to a dose-dependent suppression of anchorage-independent growth as determined by soft agar assays. A structural analog (PD159805) with no inhibitory activity against EGFR tyrosine kinase had no effect on HONE-T1 cell growth in agar. Furthermore, growth of HONE-T1 xenografts in SCID mice was also inhibited by treatment with PD158780 and PD 168393. This data provides an appealing application of EGFR tyrosine kinase inhibitors for the treatment of nasopharyngeal carcinomas.

Tyrosine kinase inhibitors. 15. 4-(Phenylamino)quinazoline and 4-(phenylamino)pyrido[d]pyrimidine acrylamides as irreversible inhibitors of the ATP binding site of the epidermal growth factor receptor.

A series of 6- and 7-acrylamide derivatives of the 4-(phenylamino)quinazoline and -pyridopyrimidine classes of epidermal growth factor receptor (EGFR) inhibitors were prepared from the corresponding amino compounds by reaction with either acryloyl chloride/base or acrylic acid/1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. All of the 6-acrylamides, but only the parent quinazoline 7-acrylamide, were irreversible inhibitors of the isolated enzyme, confirming that the former are better-positioned, when bound to the enzyme, to react with the critical cysteine-773. Quinazoline, pyrido[3,4-d]pyrimidine, and pyrido[3,2-d]pyrimidine 6-acrylamides were all irreversible inhibitors and showed similar high potencies in the enzyme assay (likely due to titration of the available enzyme). However the pyrido[3,2-d]pyrimidine analogues were 2-6-fold less potent than the others in a cellular autophosphorylation assay for EGFR in A431 cells. The quinazolines were generally less potent overall toward inhibition of heregulin-stimulated autophosphorylation of erbB2 (in MDA-MB-453-cells), whereas the pyridopyrimidines were equipotent. Selected compounds were evaluated in A431 epidermoid and H125 non-small-cell lung cancer human tumor xenografts. The compounds showed better activity when given orally than intraperitoneally. All showed significant tumor growth inhibition (stasis) over a dose range. The poor aqueous solubility of the compounds was a drawback, requiring formulation as fine particulate emulsions.

Regulation of metastasis-related gene expression by p53: a potential clinical implication.

Tumor metastasis is the main cause of mortality and treatment failure in cancer patients. It is a complex biological process regulated by alternations in expression of many genes. The p53 tumor suppressor gene has been shown to regulate expression of some metastasis-related genes. p53 transcriptionally activates expression of the genes encoding epidermal growth factor receptor, matrix metalloproteinase (MMP)-2, cathepsin D, and thrombospondin-1 but represses expression of the genes encoding basic fibroblast growth factor and multidrug resistance-1. Decreased expression of E-cadherin is associated with p53 alternations. Because these p53-regulatory genes either promote or inhibit tumor metastasis, the net effect of p53 expression on tumor metastasis depends upon the pattern of expression of these genes in a particular tumor. Because radiotherapy has been shown to increase tumor metastasis in both animal and human studies and because p53 is activated by radiation or DNA-damaging reagents, here we propose the working hypothesis that p53 may promote tumor metastasis upon induction by local radiotherapy or chemotherapy in some tumor types. For patients whose tumors contain wild-type p53, MMP inhibitors might be given with or before radiotherapy or chemotherapy to prevent an increase in tumor metastasis. Special caution should be taken with patients with cancers such as nasopharyngeal carcinoma in which p53 mutation is infrequent and radiotherapy is the main choice of treatment. To test our hypothesis, three studies are proposed and could serve as an initial step in understanding the complex biological process following radiation-induced p53 activation and its roles in regulation of tumor metastasis.

Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor.

A class of high-affinity inhibitors is disclosed that selectively target and irreversibly inactivate the epidermal growth factor receptor tyrosine kinase through specific, covalent modification of a cysteine residue present in the ATP binding pocket. A series of experiments employing MS, molecular modeling, site-directed mutagenesis, and 14C-labeling studies in viable cells unequivocally demonstrate that these compounds selectively bind to the catalytic domain of the epidermal growth factor receptor with a 1:1 stoichiometry and alkylate Cys-773. While the compounds are essentially nonreactive in solution, they are subject to rapid nucleophilic attack by this particular amino acid when bound in the ATP pocket. The molecular orientation and positioning of the acrylamide group in these inhibitors in relation to Cys-773 entirely support these results as determined from docking experiments in a homology-built molecular model of the ATP site. Evidence is also presented to indicate that the compounds interact in an analogous fashion with erbB2 but have no activity against the other receptor tyrosine kinases or intracellular tyrosine kinases that were tested in this study. Finally, a direct comparison between 6-acrylamido-4-anilinoquinazoline and an equally potent but reversible analog shows that the irreversible inhibitor has far superior in vivo antitumor activity in a human epidermoid carcinoma xenograft model with no overt toxicity at therapeutically active doses. The activity profile for this compound is prototypical of a generation of tyrosine kinase inhibitors with great promise for therapeutic significance in the treatment of proliferative disease.

Amino-terminal sequence determinants for substrate recognition by platelet-derived growth factor receptor tyrosine kinase.

The specificity of protein kinases has been shown to be influenced by residues near the phosphoaccepting amino acid. To examine the determinants for platelet-derived growth factor receptor (PDGFR) tyrosine kinase specificity, a peptide library with three degenerate positions N-terminal to tyrosine was constructed. After reaction with PDGFR, the most abundant phosphopeptides were isolated by immunoaffinity chromatography on a column containing monoclonal anti-phosphotyrosine antibody. Further separation of bound phosphopeptides with reverse-phase HPLC led to the identification of three optimal substrates for PDGFR: Ala-Ala-Asn-Ile-Thr-Tyr-Ala-Ala-Arg-Arg-Gly, Ala-Ala-Asn-Arg-Thr-Tyr-Ala-Ala-Arg-Arg-Gly and Ala-Ala-Leu-Ile-Thr-Tyr-Ala-Ala-Arg-Arg-Gly, where underlined residues are in the degenerate positions of the peptide library. Kinetic analyses of the three individual peptides (synthesized separately) showed these peptides to be among the best reported substrates for PDGFR. Our results expand the range of amino acid residues that have been shown to serve as recognition elements for receptor tyrosine kinases.

Inhibition of the epidermal growth factor receptor tyrosine kinase by PD153035 in human A431 tumors in athymic nude mice.

PD153035 is a potent (Ki = 6 pm) and specific inhibitor of the epidermal growth factor (EGF) receptor tyrosine kinase that suppresses tyrosine phosphorylation of the EGF receptor in A431 cells at nanomolar concentrations in cell culture. We have examined the pharmacokinetics of this compound and its ability to rapidly suppress phosphorylation of the EGF receptor in A431 human epidermoid tumors grown as xenografts in immunodeficient nude mice. Following a single i.p. dose of 80 mg/kg, the drug levels in the plasma and tumor rose to 50 and 22 microM within 15 minutes. While the plasma levels of PD153035 fell below 1 microM by 3 hours, in the tumors it remained at micromolar concentrations for at least 12 hours. The tyrosine phosphorylation of the EGF receptor was rapidly suppressed by 80-90% in the tumors. However receptor phosphorylation returned to control levels after 3 hours despite the continued presence of the drug at concentrations which, based on previous in vitro results, were predicted to maintain inhibition. EGF-stimulated tyrosine kinase activity in tumor extracts was decreased and recovered in parallel with the effects of PD153035 on receptor phosphorylation though the activity had reached only about half of the control activity after three hours. These results demonstrate the potential for using small molecule inhibitors to inhibit the EGF receptor tyrosine kinase in vivo, though a fair evaluation of their potential anti-cancer activity will have to wait for solutions to problems with sustained delivery which may allow us to maintain suppression of EGF receptor phosphorylation.

Cellular transport of CI-980.

CI-980, originally synthesized as a potential folate antagonist, is a tubulin-binding mitotic inhibitor currently in pediatric phase I and adult phase II clinical trials. Because of its extensive tissue distribution in animals and its favorable activity against multidrug resistant (MDR)-cells compared with other mitotic inhibitors, such as vincristine, we examined the membrane transport properties of CI-980. CI-980 accumulated rapidly in L1210 and CHO/K1 cells, reaching intracellular levels 40- and 8-fold higher, respectively, than those in the extracellular medium. Efflux was also quite rapid, but a small fraction of drug remained associated with the cells in drug-free medium. The uptake of CI-980 was not temperature or energy dependent, nor was it saturable up to an extracellular concentration of 100 microM. Inhibitors of nucleoside transport had no effect on CI-980 uptake. A cell line deficient in the transport of reduced folate was not resistant to CI-980, nor did it exhibit reduced CI-980 uptake. A 100-fold excess of the R-enantiomer inhibited CI-980 uptake by only 50%. These results are consistent with a model of CI-980 uptake involving passive diffusion followed by significant but largely reversible binding to intracellular or membrane components.

Preclinical antitumor activity of CI-994.

CI-994 [aka: acetyldinaline; PD 123654; 4-acetylamino-N-(2'aminophenyl)-benzamide] (Figure 1) is a novel antitumor agent with a unique mechanism of action. It is the acetylated metabolite of dinaline, a compound previously identified as having cytotoxic and cytostatic activity against several murine and human xenograft tumor models. CI-994 had activity against 8/8 solid tumors tested (log cell kills at the highest non-toxic dose): pancreatic ductal adenocarcinoma #02 (4.7); pancreatic adenocarcinoma #03 (3.0; 1/6 cures); colon adenocarcinoma #38 (1.6); colon adenocarcinoma #51/A (1.1); mammary adenocarcinoma #25 (1.7); mammary adenocarcinoma #17/ADR (0.5); Dunning osteogenic sarcoma (4.0); and the human prostate carcinoma LNCaP (1.2). CI-994 had the same spectrum of activity in vivo as dinaline. It also behaved similarly in schedule comparison/toxicity trials. Prolonged administration with lower drug doses was more effective than short-term therapy at higher individual doses. If doses were kept between 40 and 60 mg/kg/injection, prolonged administration (> 50 days) was tolerated with no gross toxicity. Doses > or = 90 mg/kg/injection caused lethality after 4-5 days of administration. The maximum tolerated total dose was also increased with smaller individual doses administered for prolonged intervals. Clinical Phase I trials are ongoing with this agent.

Chemotherapy with [SP-4-3-(R)]-[1,1-cyclobutanedicarboxylato(2-)](2- methyl-1,4-butanediamine-N,N')platinum (CI-973, NK121) in combination with standard agents against murine tumors in vivo.

[SP-4-3-(R)]-[1,1-cyclobutanedicarboxylato(2-)](2-methyl-1,4- butane-diamine-N,N')platinum (CI-973) is a cisplatin analogue that is currently in clinical trial. Preclinically, CI-973 retained activity against L1210, P388, K562, and human ovarian carcinoma sublines resistant to cisplatin in vitro. CI-973 also retained substantial activity [ratio of median life span of treated to control groups x 100% (%T/C) > 190] against cisplatin resistant L1210 and P388 sublines in vivo. Good activity (stasis or tumor burden reduction) was also obtained against five murine solid tumors including breast, colon, and sarcoma. Binary combination therapy with CI-973 and seven clinically utilized anticancer agents was evaluated against murine tumors in vivo for the ability of each combination to produce a superior therapeutic response compared to optimal single agent therapy alone at tolerated doses. The seven agents combined with CI-973 were mitomycin C, cyclophosphamide, doxorubicin, vinblastine, etoposide, ifosfamide, and methotrexate. Of the combination regimens evaluated, only the combination of CI-973 and methotrexate was therapeutically superior to single agent therapy. Against i.v. inoculated P388 leukemia, combination therapy with CI-973 at 32 mg/kg/injection and methotrexate at 43 mg/kg/injection produced 5.3 logs greater cell kill (%T/C = 284, with one cell surviving therapy) than either methotrexate therapy (%T/C = 208, with 2.5 x 10(5) cells surviving therapy) or CI-973 therapy (%T/C = 193 with 2.5 x 10(6) cells surviving therapy) alone. The combination toxicity index of 1.0 indicated additive normal host tissue toxicity with the same target organs for dose limiting toxicity. To better understand the enhanced cell kill in vivo, the combination of CI-973 and methotrexate was evaluated against P388 leukemia in vitro. Clonogenic survival studies showed that the combination of CI-973 and methotrexate was additive rather than synergistic with respect to cell killing in vitro. The lack of greater than additive cell kill in vitro suggested that the mechanism responsible for synergistic kill in vivo was not operative in vitro. The in vivo results suggest that the combination of CI-973 and methotrexate may be useful in the clinic.

A specific inhibitor of the epidermal growth factor receptor tyrosine kinase.

A small molecule called PD 153035 inhibited the epidermal growth factor (EGF) receptor tyrosine kinase with a 5-pM inhibition constant. The inhibitor was specific for the EGF receptor tyrosine kinase and inhibited other purified tyrosine kinases only at micromolar or higher concentrations. PD 153035 rapidly suppressed autophosphorylation of the EGF receptor at low nanomolar concentrations in fibroblasts or in human epidermoid carcinoma cells and selectively blocked EGF-mediated cellular processes including mitogenesis, early gene expression, and oncogenic transformation. PD 153035 demonstrates an increase in potency over that of other tyrosine kinase inhibitors of four to five orders of magnitude for inhibition of isolated EGF receptor tyrosine kinase and three to four orders of magnitude for inhibition of cellular phosphorylation.

Strategies for the discovery of novel tyrosine kinase inhibitors with anticancer activity.

We present a general drug discovery strategy to find novel inhibitors of tyrosine kinases. This scheme includes the production of initial protein targets for primary screening, an evaluation of enzyme potency and specificity, biochemical and biological effects on various cellular processes in whole-cell models, and strategies for in vivo evaluation of antitumor activity. These aspects are illustrated using a newly discovered class of tyrosine kinase inhibitors, the 2-thioindoles. Certain members of this class of compounds inhibit specific tyrosine kinases at low micromolar concentrations and exhibit a distinct structure-activity relationship, as well as enzyme specificity depending on the chemical substitution. These compounds suppress growth factor-mediated tyrosine phosphorylation and mitogenesis in viable cells and, in some cases, exhibit marked specificity for these effects depending on the substituents on the indole ring system. The issue is stressed that since inhibitors of signal transduction pathways represent an entirely new class of potential antitumor agents, distinct from past and currently used cancer therapies, alternative in vivo tumor models may be needed as well as different requirements for dose levels, scheduling and endpoint evaluation. These inherent difficulties emphasize a need for more basic research at the in vivo stage of drug evaluation to enhance model development and provide a better understanding of the pharmacology for these newer classes of drugs.

Comparative molecular field analysis of in vitro growth inhibition of L1210 and HCT-8 cells by some pyrazoloacridines.

In vitro screening of a number of 2-(aminoalkyl)-5-nitropyrazolo[3,4,5- kl]acridines has previously indicated (Sebolt, J.S.; et al. Cancer Res. 1987, 47, 4299-4304) that these compounds, in general, exhibit selective cytotoxicity against the human colon adenocarcinoma, HCT-8, cell line, relative to mouse leukemia L1210 cells. Comparative molecular field analysis (CoMFA) was applied to HCT-8 and L1210 growth inhibition assays (IC50s) of a series (44) of the pyrazoloacridine derivatives with the objective of predicting improved solid tumor selectivity. In the absence of crystallographic data, the 9-methoxy derivative (15), which is currently in clinical study, was selected as the template molecular model. Two different structural alignments were tested: an alignment of structures based on root mean square (RMS)-fitting of each structure to 15 was compared with an alternative strategy, steric and electrostatic alignment (SEAL). Somewhat better predictive cross-validation correlations (r2) were obtained with models based on RMS vis-à-vis SEAL alignment for both sets of assays. A large change in lattice spacing, e.g., 2 to 1 A, causes significant variations in the CoMFA results. A shift in the lattice of half of its spacing had a much smaller effect on the CoMFA data for a lattice of 1 A than one of 2 A. The relative contribution of steric and electrostatic fields to both models were about equal, underscoring the importance of both terms. Neither calculated log P nor HOMO and/or LUMO energies contribute to the model. Steric and electrostatic fields of the pyrazoloacridines are the sole relevant descriptors to the structure-activity (cross-validated and conventional) correlations obtained with the cytotoxic data for both the L1210 and HCT-8 cell lines. The cross-validated r2, derived from partial least-squares calculations, indicated considerable predictive capacity for growth inhibition of both the leukemia and solid-tumor data. Evidence for the predictive performance of the CoMFA-derived models is provided in the form of plots of actual vs predicted growth inhibition of L1210 and HCT-8 cells, respectively, by the pyrazoloacridines. The steric and electrostatic features of the QSAR are presented in the form of standard deviation coefficient contour maps of steric and electrostatic fields. The maps indicate that increases or decreases in steric bulk that would enhance growth inhibition of HCT-8 cells would likewise promote growth inhibition of L1210 cells. Contour maps generated to analyze the electrostatic field contributions of the pyrazoloacridines to growth inhibition provide an essentially similar set of results.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of 5-phosphoribosyl-1-pyrophosphate synthetase by the monophosphate metabolite of 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine: a novel mechanism for antitumor activity.

The aminopyrimidopyrimidine nucleoside 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (APP), which was previously shown to possess experimental antitumor and antiviral activity, was metabolized within WI-L2 human lymphoblastoid cells to a derivative identified as the beta-D-ribonucleotide (APP-MP). In a subline of WI-L2 cells deficient in adenosine kinase, this metabolite was not formed and APP was not cytotoxic, suggesting that APP is converted by adenosine kinase to its 5'-monophosphate. Because no evidence of di- or triphosphates was seen, the monophosphate appeared to be the active species. Treatment of WI-L2 or L1210 cells with APP (10 microM) for 30 min caused extensive depletion of both purine and pyrimidine ribonucleotides. Purine and pyrimidine deoxyribonucleotides were also depleted. Cells were not protected from the cytotoxicity of APP by hypoxanthine plus uridine, but uridine plus adenosine plus 2-deoxycoformycin gave considerable protection. This result was consistent with APP-MP acting as an inhibitor of 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase, a hypothesis that was confirmed by preparing PRPP synthetase from Novikoff hepatoma cells; APP-MP was a noncompetitive inhibitor, with a Ki of 0.43 mM. APP-MP was found to accumulate in APP-treated cells to a concentration of almost 3 mM. The relevance of PRPP synthetase inhibition to the cytotoxic mechanism of APP is indicated by the fact that depletion of the PRPP pool was seen as early as 15 min after treatment, before any change was apparent in cellular levels of ATP or UTP. DNA synthesis was markedly suppressed within 30 min of APP treatment of WI-L2 cells, and a lesser degree of inhibition of RNA synthesis was apparent after 45 min.