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.

Accumulation of cis-diamminedichloroplatinum(II) and platinum analogues by platinum-resistant murine leukemia cells in vitro.

Three murine leukemia lines resistant to cis-diamminedichloroplatinum(II) and one line resistant to diaminocyclohexane (DACH) platinum(II) complexes were compared to their platinum-sensitive parent lines to determine whether differences in net platinum accumulation were related to the resistant phenotype. The cis-diamminedichloroplatinum(II)-resistant lines (L1210PtR4, L1210DDP5, P388PtR4) and the DACH-resistant line (L1210DACH) were incubated in vitro with cis-diamminedichloroplatinum(II), [sp-4-2-(1R,2R)]-(1,2-cyclohexanediamine-N-N')dichloroplatinum(II) , [sp-4-2-(1R,2R)]-(1,2-cyclohexanediamine-N-N')[ethanedioato( 2-)- O,O']platinum(II), or diaminocyclobutanedicarboxylatoplatinum(II) and the time-dependent cellular platinum levels determined by flameless atomic absorption spectrophotometry. Cell lines resistant to a given platinum complex showed a reduction in the rate of platinum accumulation when compared to the sensitive line at 37 degrees C. Intracellular levels of diaminocyclobutanedicarboxylatoplatinum(II) were too low to confidently measure under the conditions of this study. Our data suggest that the mechanism of platinum resistance in these cell lines may be related to a reduced accumulation of the platinum-containing drug, although patterns of cross-resistance suggest other mechanisms may be operative as well.

The pyrido[2,3-d]pyrimidine derivative PD180970 inhibits p210Bcr-Abl tyrosine kinase and induces apoptosis of K562 leukemic cells.

PD180970 is a novel pyrido[2,3-d]pyrimidine class of ATP-competitive inhibitor of protein tyrosine kinases. We found that PD180970 inhibited in vivo tyrosine phosphorylation of p210Bcr-Abl (IC50 = 170 nM) and the p210BcrAbl substrates Gab2 and CrkL (IC50 = 80 nM) in human K562 chronic myelogenous leukemic cells. In vitro, PD180970 potently inhibited autophosphorylation of p210Bcr-Abl (IC50 = 5 nM) and the kinase activity of purified recombinant Abl tyrosine kinase (IC50 = 2.2 nM). Incubation of K562 cells with PD180970 resulted in cell death. Results of nuclear staining, apoptotic-specific poly(ADP-ribose) polymerase cleavage, and annexin V binding assays indicated that PD180970 induced apoptosis of K562 cells. In contrast, PD180970 had no apparent effects on the growth and viability of p210Bcr-Abl-negative HL60 human leukemic cells. Thus, PD180970 is among the most potent inhibitors of the p210Bcr-Abl tyrosine kinase, which is present in almost all cases of human chronic myelogenous leukemia. These findings indicate that PD180970 is a promising candidate as a novel therapeutic agent for Bcr-Abl-positive leukemia.

Zinc metabolism in Ehrlich cells: properties of a metallothionein-like zinc-binding protein.

Host zinc deficiency halts the proliferation of the mouse Ehrlich ascites tumor. The major site of measurable cellular zinc depletion is a cytosolic zinc binding protein. This protein is characterized as a metallothionein on the basis of its presence as two isoproteins which behave on DEAE-Sephadex and in polyacrylamide gel electrophoresis like metallothioneins, the lack of protein absorbance at 280 nm, its sulfhydryl/zinc ratio of 3.5, and its reactivity in zinc transfer to apocarbonic anhydrase. Finally, the protein exhibits cross-reactivity with a known rat metallothionein in a radioimmunoassay. Coupled with the similarity in structure and antigenicity of rat and mouse metallothioneins, this adds strong support to the identification of the zinc-binding protein as a metallothionein. In zinc-deficient cells this metallothionein-like protein appears to exist as an apoprotein. When small amounts of dietary zinc stimulate the deficient cells to divide, zinc is not observed in metallothionein. Larger concentrations of dietary zinc support both proliferation and the steady state presence of zinc in this protein. It is demonstrated that metallothionein is the principal donor of zinc to apocarbonic anhydrase added to Ehrlich cytosol. These results are used to construct a model of zinc metabolism in which zinc metallothionein is a labile depot of zinc in the Ehrlich cell which can be mobilized under zinc-deficient conditions.

Inhibition of Src kinases by a selective tyrosine kinase inhibitor causes mitotic arrest.

src kinase activity is elevated in some human tumors, including breast and colon cancers. The precise cellular function of the src family kinases is not clearly understood, but they appear to be involved in numerous signaling pathways. We studied the effects of PD173955, a novel src family-selective tyrosine kinase inhibitor, on cancer cell lines and found that it has significant antiproliferative activity due to a potent arrest of mitotic progression. The mitotic block occurs after chromosome condensation in prophase, before spindle assembly and without loss of cyclin A and B kinase activities. This effect is seen in cancer cell lines of all types with low or high activities of src kinases as well as in untransformed cell lines. In MDA-MB-468 breast cancer cells, this drug produces a rapid inhibition of cellular src and yes kinase activities as well as suppression of the mitotic hyperactivity of these kinases. This compound defines a novel class of antimitotic drugs that work through inhibition of src kinases and possibly other protein kinases that are required for progression through the initial phases of mitosis.

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.

Activation of Stat3 preassembled with platelet-derived growth factor beta receptors requires Src kinase activity.

Members of the STAT family of transcriptional regulators modulate the expression of a variety of gene products that promote cell proliferation, survival and transformation. Although initially identified as mediators of cytokine signaling, the STAT proteins are also activated by, and thus may contribute to the actions of, polypeptide growth factors. To define the mechanism by which these factors activate STATs, we examined the process of Stat3 activation in Balb/c-3T3 fibroblasts treated with platelet-derived growth factor (PDGF). As STATs are activated by tyrosine phosphorylation, and as PDGF receptors are ligand-activated tyrosine kinases, we considered the possibility that Stat3 interacts with and is phosphorylated by PDGF receptors. We find that Stat3 associates with PDGF beta receptors in both the presence and, surprisingly, the absence of PDGF. Moreover, Stat3 was phosphorylated on tyrosine in PDGF beta receptor immunoprecipitates of PDGF-treated but not untreated cells. Although required, receptor activation was insufficient for Stat3 activation. When added to cells in combination with a pharmacologic agent (PD180970) that specifically inhibits the activity of Src family tyrosine kinases, PDGF did not activate Stat3 as monitored by electrophoretic mobility shift assay. PD180970 did not affect MAPK activation by PDGF or the JAK-dependent activation of Stat3 by interleukin-6. The necessity of Src activity for Stat3 activation by PDGF was further evidenced by data showing the presence of Src in complexes containing both Stat3 and PDGF beta receptors in PDGF-treated cells. These results suggest a novel mechanism of STAT activation in which inactive Stat3 pre-assembles with inactive PDGF receptors, and in response to ligand binding and in a manner dependent on Src kinase activity, is rapidly phosphorylated and activated. Additional data demonstrate that Src kinase activity is also required for PDGF stimulation of DNA synthesis in density-arrested cells.

Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells.

Constitutive activation of signal transducer and activator of transcription (STAT) proteins has been detected in a wide variety of human primary tumor specimens and tumor cell lines including blood malignancies, head and neck cancer, and breast cancer. We have previously demonstrated a high frequency of Stat3 DNA-binding activity that is constitutively-induced by an unknown mechanism in human breast cancer cell lines possessing elevated EGF receptor (EGF-R) and c-Src kinase activities. Using tyrosine kinase selective inhibitors, we show here that Src and JAK family tyrosine kinases cooperate to mediate constitutive Stat3 activation in the absence of EGF stimulation in model human breast cancer cell lines. Inhibition of Src or JAKs results in dose-dependent suppression of Stat3 DNA-binding activity, which is accompanied by growth inhibition and induction of programmed cell death. In addition, transfection of a dominant-negative form of Stat3 leads to growth inhibition involving apoptosis of breast cancer cells. These results indicate that the biological effects of the Src and JAK tyrosine kinase inhibitors are at least partially mediated by blocking Stat3 signaling. While EGF-R kinase activity is not required for constitutive Stat3 activation in breast cancer cells, EGF stimulation further increases STAT DNA-binding activity, consistent with an important role for EGF-R in STAT signaling and malignant progression. Analysis of primary breast tumor specimens from patients with advanced disease revealed that the majority exhibit elevated STAT DNA-binding activity compared to adjacent non-tumor tissues. Our findings, taken together, suggest that tyrosine kinases transduce signals through Stat3 protein that contribute to the growth and survival of human breast cancer cells in culture and potentially in vivo.

Small molecule inhibitors of the platelet-derived growth factor receptor, the fibroblast growth factor receptor, and Src family tyrosine kinases.

The inhibition of tyrosine kinases involved in growth factor signal transduction pathways represents an attractive strategy for controlling aberrant cellular growth. Over the last 4-5 years, there have been numerous reports on the discovery of small molecule inhibitors for potential therapeutic applications to a number of proliferative diseases, principally cancer and restenosis, where the over-expression of certain tyrosine kinases has been demonstrated. These include, amongst others, the platelet-derived growth factor receptor, the fibroblast growth factor receptor, and the nonreceptor c-Src tyrosine kinase. This review compiles published reports and patent filings from 1995 to mid-1997 that include data directly related to inhibition of the platelet-derived growth factor receptor, fibroblast growth factor receptor, and Src family tyrosine kinases. Potential clinical applications for selected classes of tyrosine kinase inhibitors reviewed herein will likely depend on the demonstration of meaningful activity in a variety of therapeutic targets in animal models.

Therapeutic targeting of Src-kinase Lyn in myeloid leukemic cell growth.

Protein tyrosine kinases play a major role in promoting cell growth, and their activity in solid tumors is well established. Inhibitors of protein tyrosine kinases are now in advanced clinical trials for the treatment of breast and brain cancers. Because Src-related PTK have been shown to be activated in leukemic cell lines, we studied their activation in human myeloid leukemia. Blasts from the majority of patients with acute leukemia showed constitutive activity of the Src kinase Lyn. In contrast, no patient samples showed constitutive activation of Jak2. Genetic and pharmacologic targeting of Lyn was used to determine its contribution to leukemic cell growth. Antisense Lyn oligonucleotide treatment resulted in the inhibition of tritiated thymidine incorporation following GM-CSF stimulation of the factor-dependent line MO7e. The Src kinase inhibitor PD166285 inhibited the growth of human leukemic cell lines and leukemic blasts. When combined with doxorubicin, an additive effect on the inhibition of leukemic cell growth occurred. These studies demonstrate the importance of Src kinases in promoting leukemic cell growth and suggests that further development of agents which target Src kinases and their inclusion in multidrug regimens are warranted for novel therapies of myeloid leukemia.

Interleukin-3 protects Bcr-Abl-transformed hematopoietic progenitor cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors.

Bcr-Abl tyrosine kinase has been validated as a molecular target for the treatment of chronic myelogenous leukemia (CML). More recently, it has been reported that CML patients could develop resistance to the Bcr-Abl tyrosine kinase inhibitor, imatinib (STI571, Gleevec), pointing to the need for development of additional Bcr-Abl tyrosine kinase inhibitors or other therapeutic strategies. It was also found that a significant proportion of patients who received the Bcr-Abl inhibitor did not achieve complete cytogenetic response. Mechanisms for incomplete cytogenetic response to Bcr-Abl inhibition are not entirely clear. We report here three new pyrido[2,3-d]pyrimidine Bcr-Abl tyrosine kinase inhibitors, PD164199, PD173952, PD173958, that induced apoptosis of Bcr-Abl-dependent hematopoietic cells. An interleukin-3 (IL-3) autocrine loop was observed previously in primitive CD34(+)/Bcr-Abl(+) leukemic cells in CML patients. Using 32Dp210(Bcr-Abl)and Baf3p210(Bcr-Abl) cells as models, we tested whether IL-3 might protect Bcr-Abltransformed, IL-3-responsive cells from apoptosis caused by Bcr-Abl tyrosine kinase inhibition. Results of trypan blue exclusion, fluoroisothiocyanate-valyl-alanyl-aspartyl-[O-methyl] -fluoromethylketone (FITC-VAD-FMK), and Annexin-V/7-amino-actinomycin D (7-AAD) binding assays indicate that IL-3 could protect Bcr-Abl-transformed, IL-3 responsive hematopoietic progenitor cells from apoptosis induced by Bcr-Abl tyrosine kinase inhibitors. This finding raises the possibility that the IL-3 autocrine loop found in primitive CD34(+)/Bcr-Abl(+) cells in CML patients could contribute to the incomplete eradication of Bcr-Abl(+) cells by Bcr-Abl inhibition.

Synthesis and biochemical evaluation of a series of aminoflavones as potential inhibitors of protein-tyrosine kinases p56lck, EGFr, and p60v-src.

A series of nitroflavones, 8a-p, and their corresponding aminoflavone hydrochloride salts, 10a-p, was synthesized. The preparation of nitroflavones 8b-i,o,p began with commercially available o-hydroxyacetophenones 2b-f which were converted to o-hydroxynitroacetophenones 3a-h via a variety of nitration methods, followed by condensation with nitrobenzoyl chlorides and cyclization under acidic condition. The nitroflavones 8aj-n were prepared by nitration of the corresponding flavones 7a-e. These new compounds were evaluated for their abilities to inhibit the in vitro protein-tyrosine kinase activities of p56lck, EGFr, and p60v-src, and all of the active compounds were amino-substituted flavones. None of the nitroflavones inhibited the enzymes. The most active substance in this series against p56lck was compound 10j, which had an IC50 of 18 microM. When tested versus EGFr, compounds 10a,m displayed IC50's of 8.7 and 7.8 microM, respectively. Against p60v-src, 10a,m showed IC50 values of 28.8 and 38.4 microM, respectively.

Tyrosine kinase inhibitors. 7. 7-Amino-4-(phenylamino)- and 7-amino-4-[(phenylmethyl)amino]pyrido[4,3-d]pyrimidines: a new class of inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor.

The synthesis of 7-aminopyrido[4,3-d]pyrimidines bearing aromatic side chains at the 4-position is reported. These compounds are shown to be a new class of inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR). Structure-activity relationships (SARs) for substitution in both 4-(phenylamino)- and 4-[(phenylmethyl)amino] side chains were determined, using a series of substituents (NO2, Br, CF3, OMe, NH2, and NMe2) selected primarily for their wide range of electronic properties. In the phenylamino series, 3-substituted derivatives were more potent than the corresponding 2- and 4-substituted analogues. For the 3-substituted compounds, activity was favored by electron withdrawal, in a relationship which could be quantified, with the 3-Br being the most potent compound (IC50 = 0.01 microM compared with IC50 = 0.34 microM for the unsubstituted side chain derivative). No such correlation was apparent for the 2- or 4-substituent, although Br was still the best substituent. In contrast, in the 4-[(phenylmethyl)amino] series, substitution of the side chain was not beneficial. For the 4-(phenylamino) series, the substituent SARs are broadly similar to that found previously for 4-(phenylamino)quinazolines. These results suggest that side chain SARs may be broadly invariant over a range of different chromophores, with the side chain of choice for optimization of EGFR inhibitory activity being 4-[(3-bromophenyl)amino].

Tyrosine kinase inhibitors. 2. Synthesis of 2,2'-dithiobis(1H-indole-3-alkanamides) and investigation of their inhibitory activity against epidermal growth factor receptor and pp60v-src protein tyrosine kinases.

A series of amide analogues of the 2,2'-dithiobis(1H-indole-3-alkaonic acid) class of tyrosine kinase inhibitors have been prepared, by reaction of 1H-indole-3-alkanamides (8) with S2Cl2, and separation of the desired disulfides from the initial mixtures of mono-, di-, and trisulfides formed. These amides were evaluated in vitro against epidermal growth factor receptor and pp60v-src protein tyrosine kinases. Inhibitory activity against EGF receptor tyrosine kinase was chain-length dependent, with the propanamides being the most effective. Hydrogen bond donor capabilities in the amide function did not appear to be necessary, with an N-benzylamide being the most potent (IC50 = 0.85 microM). Further substitution on the benzyl ring did not increase potency, and substitution in the alpha-position of the propanamide side chain was acceptable. A water-soluble alpha-NH2 derivative showed good inhibitory activity toward the enzyme, was a potent inhibitor of cell growth in fibroblasts, and selectively inhibited intracellular tyrosine phosphorylation patterns. The nonreceptor kinase pp60v-src was in general much more sensitive than EGF receptor kinase to inhibition by these compounds, but with less pronounced structure-activity relationships.

Tyrosine kinase inhibitors. 8. An unusually steep structure-activity relationship for analogues of 4-(3-bromoanilino)-6,7-dimethoxyquinazoline (PD 153035), a potent inhibitor of the epidermal growth factor receptor.

4-(3-Bromoanilino)-6,7-dimethoxyquinazoline (32, PD 153035) is a very potent inhibitor (IC50 0.025 nM) of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR), binding competitively at the ATP site. Structure-activity relationships for close analogues of 32 are very steep. Some derivatives have IC50s up to 80-fold better than predicted from simple additive binding energy arguments, yet analogues possessing combinations of similar phenyl and quinazoline substituents do not show this "supra-additive" effect. Because some substituents which are mildly deactivating by themselves can be strongly activating when used in the correct combinations, it is proposed that certain substituted analogues possess the ability to induce a change in the conformation of the receptor when they bind. There is some bulk tolerance for substitution in the 6- and 7-positions of the quinazoline, so that 32 is not the optimal inhibitor for the induced conformation. The diethoxy derivative 56 [4-(3-bromoanilino)-6,7-diethoxyquinazoline] shows an IC50 of 0.006 nM, making it the most potent inhibitor of the tyrosine kinase activity of the EGFR yet reported.

Tyrosine kinase inhibitors. 3. Structure-activity relationships for inhibition of protein tyrosine kinases by nuclear-substituted derivatives of 2,2'-dithiobis(1-methyl-N-phenyl-1H-indole-3-carboxamide).

A series of indole-substituted 2,2'-dithiobis(1-methyl-N-phenyl-1H-indole-3-carboxamides) were prepared and evaluated for their ability to inhibit the tyrosine kinase activity of both the epidermal growth factor receptor (EGFR) and the nonreceptor pp60v-src tyrosine kinase. The compounds were synthesized by conversion of appropriate 1-methyloxindoles to 1-methyl-2-indolinethiones with P2S5 followed by subsequent reaction with NaH and phenyl isocyanate and oxidative dimerization of the resulting 2,3-dihydro-N-phenyl-2-thioxo-1H-indole-3-carboxamides. The parent compound and many of the substituted analogues were moderately potent inhibitors of both kinase enzymes, but no clear relationships were seen between substitution on the indole ring and inhibitory activity. While 4-substituted compounds were generally inactive, 5-substituted derivatives with electron-withdrawing groups showed inhibitory activity. However, none of the substituted compounds showed significantly better activity than the unsubstituted parent compound. There was generally a good correlation between activity against the EGFR and pp60v-src kinases, but several compounds did show some specificity (> 20-fold) of inhibition; 5-Cl and 5-Br derivatives preferentially inhibited pp60v-src, while the 5-CF3 compound preferentially inhibited EGFR. Selected compounds from the series were found to inhibit the growth of Swiss 3T3 fibroblasts with IC50S in the range 2-25 microM, the most active being 4-substituted derivatives. The compounds inhibited bFGF-mediated protein tyrosine phosphorylation in intact cells more effectively than EGFR- or PDGF-mediated phosphorylation.

Synthesis, structural characterization, and antitumor properties of a novel class of large-ring platinum(II) chelate complexes incorporating the cis-1,4-diaminocyclohexane ligand in a unique locked boat conformation.

The first two analogs 5a,b of a new class of neutral large-ring square-planar Pt(II) chelate complexes of the generic structure [Pt(cis-1,4-dach)X2] were synthesized via a refined technique, structurally characterized by NMR (1H, 13C, 195Pt), FAB mass spectrometry, and X-ray crystallography, and evaluated for antitumor activity in vitro and in vivo in sensitive and Pt-resistant murine leukemia cell systems. An X-ray crystal structure analysis confirmed that [Pt(cis-1,4-dach)malonate] 5b is monomeric and that the cis-1,4-diaminocyclohexane (dach) ligand is incorporated in a unique and previously unknown locked boat conformation. Complex 5b crystallized as colorless rectangular plates in the orthorhombic space group Pcmn with Z = 4 and the lattice parameters a = 6.239(1) A, b = 9.965(2) A, and c = 18.437(4) A. Important structural parameters are Pt-O = 2.024(5) A, Pt-N = 2.021(6) A, N-Pt-N = 100 degrees, and N-Pt-O = 85 degrees; R = 0.0515, Rw = 0.0635. Antitumor results in murine tumor models show that the parent molecule 5a (X2 = 2 Cl) (a) is more dose potent than cisplatin against the leukemias and solid tumors examined, (b) possesses significant activity against cisplatin-resistant leukemias, but exhibits partial cross-resistance with cisplatin, and (c) may possess a spectrum of activity different from that of cisplatin. Antitumor test results in vitro indicate that (a) 5a is at least equivalent to cisplatin in dose potency and effectiveness in the leukemia cell systems studied except in the [Pt(1,2-dach)Cl2]-resistant L1210 cell line, (b) the cisplatin-resistant leukemia cell systems exhibit partial cross-resistance to 5a, (c) 5a possesses either comparable or greater cytotoxicity than the reference complexes, CI-973 (3) and bis(platinum) complex 4, and (d) 5a is more effective (approximately 18-fold) than [Pt(1R,2R-dach)Cl2] 2 in inhibiting growth in the Pt(1,2-dach)-resistant L1210 cell line, suggesting that [Pt(cis-1,4-dach)Cl2] is either not recognized as or is not acting as a "typical" Pt(dach) complex. The encouraging antitumor activity of 5a, coupled with a 10-fold higher aqueous solubility compared to [Pt(1R,2R-dach)-Cl2] 2 warrants the following future studies: synthesis of selected analogs, elucidating the nature of Pt-DNA binding sites, the mechanism of action, and the mechanistic basis for the lack of cross-resistance of [Pt(cis-1,4-dach)Cl2] against the [Pt(1,2-dach)Cl2]-resistant L1210 cell line.

Tyrosine kinase inhibitors. 1. Structure-activity relationships for inhibition of epidermal growth factor receptor tyrosine kinase activity by 2,3-dihydro-2-thioxo-1H-indole-3-alkanoic acids and 2,2'-dithiobis(1H-indole-3-alkanoic acids).

A series of 2,3-dihydro-2-thioxo-1H-indole-3-alkanoic acids, and their methyl esters were prepared, the majority by oxidation of 1H-indole-3-alkanoic acids (DMSO/HCl), followed by thiation of the corresponding 2,3-dihydro-2-oxo-1H-indole-3-alkanoic acid esters. The monomeric thiones undergo facile and reversible oxidation to the corresponding 2,2'-dithiobis(1H-indole-3-alkanoic acids). The compounds were evaluated for their abilities to inhibit the tyrosine kinase activity of the epidermal growth factor receptor using a native complex contained in plasma membrane vesicles shed from cultured A431 cells, and to inhibit the growth of Swiss 3T3 mouse fibroblast in culture. Enzyme inhibitory activity is dependent on the length of the side chain, with propanoic acid derivatives showing the highest activity. The acids are generally significantly more potent than the corresponding esters, and the disulfides more active than the corresponding monomers. An ability to undergo the thione-thiol tautomerism necessary for dimerization is essential, with 3,3-disubstituted compounds being inactive. Overall, the data suggest that the disulfide is the more active form, with much of the activity of the monomeric thiones being due to varying degrees of conversion to the disulfide during the assay. In the growth inhibition assay, the methyl esters are more potent than their corresponding carboxylic acids, and the dimers are generally more potent than the monomers. The data show these compounds to be a novel and potent class of inhibitors of epidermal growth factor receptor tyrosine kinase activity.

Anticancer activity in murine and human tumor cell lines of bis(platinum) complexes incorporating straight-chain aliphatic diamine linker groups.

The biological activity of a series of dinuclear bis(platinum) complexes of formula [(cis-PtX2-(NH3)]2(NH2(CH2)nNH2)] (X = Cl, n = 4-9, compounds 6-11; X2 = malonate, n = 5 or 6, compounds 12 and 13) is described in selected murine leukemia, murine solid tumor, and human tumor cell lines and in murine leukemia cell lines rendered resistant to cisplatin (cis-[Pt(NH3)2Cl2]). The bis(platinum) compounds showed greater activity in vitro against murine tumor cell lines resistant to either cisplatin or DACH ([Pt(DACH)Cl2]). The resistance factor is dependent on chain length of the diamine, and the structural feature of a dinuclear complex is of general use in reducing cross-resistance with cisplatin. In vivo [(cis-PtCl2(NH3)]2(NH2(CH2)5NH2)] (7) showed a % T/C of 204 against murine L1210 leukemia resistant to cisplatin compared to a % T/C of 104 for cisplatin itself at optimal doses. The complex [(Pt(mal)(NH3)]2(NH2(CH2)6NH2)] (13) was highly active in the colon 26 tumor line with 3/10 tumor-free survivors (dose of 186 mg/kg, ip D1,5,9); however, 13 was subject to substantial cross-resistance in the cisplatin resistant L1210 leukemia (% T/C 139 versus % T/C of 223 in the sensitive line). In four selected human tumor lines in vitro, compounds 6-11 were uniformly more potent than cisplatin. In the corresponding xenografts, compound 7 showed greater activity in the HCT-8 (coloadenocarcinoma) and H23 (nonsmall cell lung), but diminished potency in AH125 and H520 (both nonsmall cell lung) lines in comparison to cisplatin. Retention of activity against cisplatin-resistant cell lines and a different spectrum of activity compared to cisplatin in some human tumor cell lines suggest that this class of complexes is mechanistically different from mononuclear complexes and worthy of further development toward clinical trials.

Synthesis and structure-activity relationships of 7-substituted 3-(2, 6-dichlorophenyl)-1,6-naphthyridin-2(1H)-ones as selective inhibitors of pp60(c-src).

7-substituted 3-(2,6-dichlorophenyl)-1,6-naphthyridin-2(1H)-ones are potent inhibitors of protein tyrosine kinases, with some selectivity for c-Src. The compounds were prepared by condensing 4, 6-diaminonicotinaldehyde with 2,6-dichlorophenylacetonitrile and selectively converting the 2- and 7-amino groups of the product to hydroxy and fluoro groups, respectively, by prolonged diazotization in 50% aqueous fluoboric acid. N-Methylation, followed by treatment with aliphatic diamines, aromatic amines, or their derived lithium anions, gave the desired compounds. Selected isomeric 1, 8-naphthyridin-2(1H)-ones were also prepared in order to evaluate the relative contributions of both ring A aza atoms of the related pyrido[2,3-d]pyrimidin-7(8H)-ones to the inhibitory activity. The compounds were evaluated for their ability to prevent phosphorylation of a model substrate by c-Src, FGF-1 receptor, and PDGF-beta receptor enzymes. Overall, there was a high degree of correlation of the activities against the different kinases, with c-Src being generally the most sensitive to structural changes. 1, 6-Naphthyridin-2(1H)-one analogues bearing basic aliphatic side chains [7-NH(CH(2))(n)()NRR, 7-NHPhO(CH(2))(n)()NRR, or 7-NHPhN(CH(2))(4)NMe] were the most potent against c-Src (IC(50)s of 10-80 nM), showing good selectivity with respect to PDGFR (10-300-fold) but less with respect to FGFR. The 1, 6-naphthyridin-2(1H)-ones showed broadly similar activity to the analogous pyrido[2,3-d]pyrimidin-7(8H)-ones, whereas the 1, 8-naphthyridin-2(1H)-ones were at least 10(3)-fold less potent. These results, indicating that the 3-aza atom in the pyrido[2, 3-d]pyrimidin-7(8H)-ones is mandatory, whereas the 1-aza atom is not, support the published binding model for these compounds to c-Src (J. Med. Chem. 1998, 41, 1752), where the 3-aza and 2-NH atoms form a bidentate H-bond donor-acceptor motif that interacts with Met341 and the 1-aza atom is not involved in specific binding interactions.