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1.
J Med Chem ; 43(16): 3134-47, 2000 Aug 10.
Article in English | MEDLINE | ID: mdl-10956222

ABSTRACT

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.


Subject(s)
Enzyme Inhibitors/chemical synthesis , Naphthyridines/chemical synthesis , Proto-Oncogene Proteins pp60(c-src)/antagonists & inhibitors , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Division/drug effects , Cells, Cultured , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Mice , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Naphthyridines/chemistry , Naphthyridines/pharmacology , Phosphorylation , Proto-Oncogene Proteins pp60(c-src)/chemistry , Proto-Oncogene Proteins pp60(c-src)/metabolism , Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, Fibroblast Growth Factor, Type 1 , Receptor, Platelet-Derived Growth Factor beta/chemistry , Receptor, Platelet-Derived Growth Factor beta/metabolism , Receptors, Fibroblast Growth Factor/chemistry , Receptors, Fibroblast Growth Factor/metabolism , Structure-Activity Relationship , Tumor Cells, Cultured
2.
J Med Chem ; 43(7): 1350-8, 2000 Apr 06.
Article in English | MEDLINE | ID: mdl-10753472

ABSTRACT

Ring-substituted bis(phenazine-1-carboxamides), linked by a -(CH(2))(3)NMe(CH(2))(3)- chain, were prepared from the corresponding substituted phenazine-1-carboxylic acids by reaction of the intermediate imidazolides with bis(3-aminopropyl)methylamine. The compounds were evaluated for growth inhibitory activity in a panel of tumor cell lines, including P388 leukemia, Lewis lung carcinoma, and wild-type (JL(C)) and mutant (JL(A) and JL(D)) forms of human Jurkat leukemia. The latter mutant lines are resistant to topoisomerase (topo) II targeted agents because of lower levels of the enzyme. Analogues with small, lipophilic substituents (e.g., Me, Cl) at the 9-position were the most potent inhibitors, superior to the corresponding dimeric bis(acridine-4-carboxamides) (bis-DACA analogues). Several of the compounds were preferentially (up to 2-fold) more cytotoxic toward the mutant Jurkat lines than the wild-type. To test whether this selectivity was related to topoisomerase action, the most potent of the compounds (9-methyl) was evaluated in a cell-free system. It poisoned topo I at drug concentrations of 0.25 and 0.5 microM and inhibited the catalytic activity of both topo I and topo II at concentrations of 1 and 5 microM, respectively. Results from the NCI human tumor cell line panel showed the compounds had preferential activity toward colon tumor lines (on average 9.5-fold more active in the HT29 line than in the cell line panel as a whole). Several analogues produced significant growth delays in the relatively refractory subcutaneous colon 38 tumor model in vivo. In particular, the 9-methyl compound was substantially more potent in this tumor model than the clinical dual topo I/II poison DACA (total dose 90 versus 400 mg/kg) with comparable activity. The bis(phenazine-1-carboxamides) are a new and interesting class of dual topo I/II-directed anticancer drugs.


Subject(s)
Amides/chemistry , Antineoplastic Agents/chemistry , Enzyme Inhibitors/chemistry , Phenazines/chemistry , Topoisomerase I Inhibitors , Topoisomerase II Inhibitors , Amides/chemical synthesis , Amides/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Humans , Mice , Neoplasm Transplantation , Phenazines/chemical synthesis , Phenazines/pharmacology , Structure-Activity Relationship , Tumor Cells, Cultured
3.
J Med Chem ; 43(7): 1380-97, 2000 Apr 06.
Article in English | MEDLINE | ID: mdl-10753475

ABSTRACT

4-Anilinoquinazoline- and 4-anilinopyrido[3,2-d]pyrimidine-6-acrylamides substituted with solubilizing 7-alkylamine or 7-alkoxyamine side chains were prepared by reaction of the corresponding 6-amines with acrylic acid or acrylic acid anhydrides. In the pyrido[3,2-d]pyrimidine series, the intermediate 6-amino-7-alkylamines were prepared from 7-bromo-6-fluoropyrido[3,2-d]pyrimidine via Stille coupling with the appropriate stannane under palladium(0) catalysis. This proved a versatile method for the introduction of cationic solubilizing side chains. The compounds were evaluated for their inhibition of phosphorylation of the isolated EGFR enzyme and for inhibition of EGF-stimulated autophosphorylation of EGFR in A431 cells and of heregulin-stimulated autophosphorylation of erbB2 in MDA-MB 453 cells. Quinazoline analogues with 7-alkoxyamine solubilizing groups were potent irreversible inhibitors of the isolated EGFR enzyme, with IC(50[app]) values from 2 to 4 nM, and potently inhibited both EGFR and erbB2 autophosphorylation in cells. 7-Alkylamino- and 7-alkoxyaminopyrido[3,2-d]pyrimidines were also irreversible inhibitors with equal or superior potency against the isolated enzyme but were less effective in the cellular autophosphorylation assays. Both quinazoline- and pyrido[3,2-d]pyrimidine-6-acrylamides bound at the ATP site alkylating cysteine 773, as shown by electrospray ionization mass spectrometry, and had similar rates of absorptive and secretory transport in Caco-2 cells. A comparison of two 7-propoxymorpholide analogues showed that the pyrido[3,2-d]pyrimidine-6-acrylamide had greater amide instability and higher acrylamide reactivity, being converted to glutathione adducts in cells more rapidly than the corresponding quinazoline. This difference may contribute to the observed lower cellular potency of the pyrido[3,2-d]pyrimidine-6-acrylamides. Selected compounds showed high in vivo activity against A431 xenografts on oral dosing, with the quinazolines being superior to the pyrido[3,2-d]pyrimidines. Overall, the quinazolines proved superior to previous analogues in terms of aqueous solubility, potency, and in vivo antitumor activity, and one example (CI 1033) has been selected for clinical evaluation.


Subject(s)
Acrylamides/chemical synthesis , Enzyme Inhibitors/chemical synthesis , ErbB Receptors/antagonists & inhibitors , Morpholines/chemical synthesis , Pyrimidines/chemical synthesis , Quinazolines/chemical synthesis , Acrylamides/chemistry , Acrylamides/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Biological Transport/drug effects , Caco-2 Cells , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , ErbB Receptors/metabolism , Humans , Mice , Mice, Nude , Morpholines/chemistry , Morpholines/pharmacology , Neoplasm Transplantation , Phosphorylation , Pyrimidines/chemistry , Pyrimidines/pharmacology , Quinazolines/chemistry , Quinazolines/pharmacology , Receptor, ErbB-2/metabolism , Solubility , Spectrometry, Mass, Electrospray Ionization , Transplantation, Heterologous , Tumor Cells, Cultured
4.
J Med Chem ; 42(10): 1803-15, 1999 May 20.
Article in English | MEDLINE | ID: mdl-10346932

ABSTRACT

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.


Subject(s)
Acrylamides/chemical synthesis , Adenosine Triphosphate/metabolism , Antineoplastic Agents/chemical synthesis , Enzyme Inhibitors/chemical synthesis , ErbB Receptors/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/chemical synthesis , Quinazolines/chemical synthesis , Acrylamides/chemistry , Acrylamides/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Binding Sites , Cell Line , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , ErbB Receptors/metabolism , Humans , Mice , Mice, Nude , Neoplasm Transplantation , Phosphorylation , Pyrimidines/chemistry , Pyrimidines/pharmacology , Quinazolines/chemistry , Quinazolines/pharmacology , Structure-Activity Relationship , Transplantation, Heterologous
5.
Cancer Chemother Pharmacol ; 43(4): 323-30, 1999.
Article in English | MEDLINE | ID: mdl-10071984

ABSTRACT

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), an experimental antitumour agent currently undergoing phase I clinical trial, has a maximum tolerated dose (MTD) in male BDF1 mice of 99 micromol/kg. We have found the male Sprague-Dawley rat and the New Zealand White rabbit to have greater tolerance to DMXAA, with MTDs being 990 and 330 micromol/kg, respectively. To investigate the causes of this difference, we measured plasma and urine DMXAA concentrations by high-performance liquid chromatography (HPLC) after single i.v. bolus injections of 99 and 990 micromol/kg in the rat and following a bolus dose of 99 micromol/kg and a 10-min infusion of 330 micromol/kg in the rabbit. Following administration of DMXAA at the MTD in the mouse, rat and rabbit the maximal concentrations were 600, 2,200 and 1,708 microM, respectively, whereas areas under the concentration-time curves were 2,400, 19,000 and 2,400 microMh, respectively, for unchanged DMXAA. Data obtained for mice and rabbits were satisfactorily fitted to a two-compartment model with Michaelis-Menten kinetics. DMXAA was highly bound to plasma proteins, with the highest degree of binding being found in the rabbit. A small proportion of the total dose (7.8%, 0.6% and 12.4%, respectively) was excreted unchanged in urine over 24 h. This proportion increased (to 11.6%, 3.5% and 72.4%, respectively) following alkaline hydrolysis, suggesting the presence of glucuronide metabolites. Examination of rat and mouse urine by HPLC revealed the presence of two metabolites, which were characterized by mass spectrometry and nuclear magnetic resonance to be the acyl glucuronide of DMXAA and 6-(hydroxymethyl)-5-methylxanthenone-4-acetic acid. Thus, both mice and rats metabolise DMXAA by similar pathways. The results demonstrate considerable interspecies variations in tolerance to DMXAA that cannot be explained by differences in pharmacokinetics.


Subject(s)
Antineoplastic Agents/pharmacokinetics , Xanthenes/pharmacokinetics , Xanthones , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/urine , Blood Proteins/metabolism , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Infusions, Intravenous , Injections, Intravenous , Male , Metabolic Clearance Rate , Mice , Protein Binding , Rabbits , Rats , Rats, Sprague-Dawley , Species Specificity , Time Factors , Xanthenes/blood , Xanthenes/urine
6.
Eur J Cancer ; 34(7): 1086-90, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9849459

ABSTRACT

The epidermal growth factor receptor (EGFR) is thought to mediate the action of the mitogens EGF and tumour growth factor-alpha (TGF-alpha) in a variety of cancers, including those of the lung, breast and ovary. A number of new selective inhibitors of EGFR tyrosine kinase have now been developed as potential new antitumour agents. We used a potent inhibitor of this tyrosine kinase, 6-amino-4-[(3-bromophenyl)amino]-7-(methylamino)quinazoline (SN 25531; PD 156273), to determine the responses of primary cultures derived from patients with cancer of the lung, ovary, breast, cervix and endometrium. Cells were cultured in 96-well plates and proliferation assessed by incorporation of 3H-thymidine. Measured growth inhibitory concentrations IC50 values) varied from 1 nM to 14 microM with a 1000-fold differential between sensitive and resistant cultures. Results were compared with rates of proliferation, estimated using a paclitaxel-based method. We also measured the IC50 values for the tyrosine kinase inhibitor using a number of established human cell lines, and compared them with EGFR content using fluorescent antibody staining and flow cytometry. The presence of EGFR was found to be necessary, but not sufficient, for in vitro response. Only a small number of cell lines (3 of 7 for lung, 1 of 7 for ovarian, 2 of 3 squamous cell and 0 of 12 for melanoma) were sensitive to the tyrosine kinase inhibitor. In contrast, 40 of the 50 primary cultures (including 14 of 15 lung cancer samples and 14 of 19 ovarian cancer samples) were sensitive.


Subject(s)
Enzyme Inhibitors/pharmacology , ErbB Receptors/antagonists & inhibitors , Neoplasms/drug therapy , Protein-Tyrosine Kinases/antagonists & inhibitors , Adult , Aged , Aged, 80 and over , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Division , Endometrial Neoplasms/drug therapy , Endometrial Neoplasms/pathology , Female , Flow Cytometry , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Male , Middle Aged , Neoplasms/pathology , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Tumor Cells, Cultured , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/pathology
7.
J Med Chem ; 41(5): 742-51, 1998 Feb 26.
Article in English | MEDLINE | ID: mdl-9513602

ABSTRACT

The 4-[(3-bromophenyl)amino]pyrido[3,4-d]pyrimidine PD 158780 is a very potent in vitro inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) (IC50 0.08 nM), and other members of the erbB family, by competitive binding at the ATP site of these signal transduction enzymes. A series of analogues of PD 158780 bearing solubilizing functions off the 6-methylamino substituent were prepared by reaction of the 6-fluoro derivatives with appropriate amine nucleophiles. These were evaluated for their ability to inhibit the tyrosine phosphorylating action of EGF-stimulated full-length EGFR enzyme and for inhibition of autophosphorylation of the EGFR in A431 human epidermoid carcinoma cells in culture. The most effective analogues were those bearing weakly basic substituents through a secondary amine linkage, which proved water-soluble (> 10 mM) and potent (IC50S generally < 1 nM). No clear SAR could be discerned for these compounds with respect to amine base strength or the distance of the cationic center from the chromophore, suggesting that 6-substituents are in a favorable area of bulk tolerance in the enzyme binding site. More distinct SAR emerged for the ability of the compounds to inhibit EGFR autophosphorylation in A431 cells, where analogues bearing lipophilic weak bases were preferred. Representative analogues were evaluated for antitumor effectiveness against four in vivo tumor models. Significant in vivo activity was observed in estrogen-dependent MCF-7 breast and A431 epidermoid tumors. Marginal activity was seen in an EGFR-transfected tumor model, suggesting that while this cell line requires EGF for clone formation in soft agar, other growth factors may be able to replace EGF in vivo. Also, no activity was seen against the SK-OV-3 ovarian cancer model, which is known to express other EGF receptor family members (although it is not clear whether these are absolutely required for growth in vivo). While substantial growth delays were seen in A431 and MCF-7 tumor models, the treated tumors remained approximately the same size throughout therapy, suggesting that the compounds are cytostatic rather than cytotoxic under these test conditions. It remains to be determined if more prolonged therapy has cytotoxic effects in vivo, resulting in net tumor cell kill.


Subject(s)
Antineoplastic Agents/chemical synthesis , Enzyme Inhibitors/chemical synthesis , ErbB Receptors/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/chemistry , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , 3T3 Cells , Animals , Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/metabolism , Enzyme Inhibitors/pharmacology , ErbB Receptors/genetics , Female , Humans , Mice , Mice, Nude , Molecular Structure , Ovarian Neoplasms/drug therapy , Phosphorylation , Pyrimidines/pharmacology , Structure-Activity Relationship , Transfection , Tumor Cells, Cultured
8.
Biochem Pharmacol ; 54(8): 877-87, 1997 Oct 15.
Article in English | MEDLINE | ID: mdl-9354588

ABSTRACT

The tyrosine kinase inhibitors PD 69896, 153717, and 158780, which belong to the chemical class 4-[ar(alk)ylamino]pyridopyrimidines, have been characterized with respect to enzymology, target specificity, and antiproliferative effects in tumor cells. These compounds were competitive inhibitors with respect to ATP against purified epidermal growth factor (EGF) receptor tyrosine kinase and inhibited EGF receptor autophosphorylation in A431 human epidermoid carcinoma with IC50 values of 2085, 110, and 13 nM, respectively. Onset of inhibition was immediate once cells were exposed to these compounds, whereas recovery of receptor autophosphorylation activity after the cells were washed free of the compound was dependent on inhibitory potency. Thus, full activity returned immediately after removal of PD 69896 but required 8 hr after exposure to PD 158780. PD 158780 was highly specific for the EGF receptor in Swiss 3T3 fibroblasts, inhibiting EGF-dependent receptor autophosphorylation and thymidine incorporation at low nanomolar concentrations while requiring micromolar levels for platelet-derived growth factor- and basic fibroblast growth factor-dependent processes. PD 158780 inhibited heregulin-stimulated phosphorylation in the SK-BR-3 and MDA-MB-453 breast carcinomas with IC50 values of 49 and 52 nM, respectively, suggesting that the compound was active against other members of the EGF receptor family. The antiproliferative effects of this series of compounds against A431 cells correlated precisely with the inhibitory potency against EGF receptor autophosphorylation. PD 158780 reduced clone formation in soft agar of fibroblasts transformed by EGF, EGF receptor, or the neu oncogene but not ras or raf, further demonstrating its high degree of specificity. Finally, this compound was active against clone formation in several breast tumors having different expression patterns of the erbB family, indicating an anticancer utility in tumors expressing these receptors.


Subject(s)
Enzyme Inhibitors/pharmacology , ErbB Receptors/antagonists & inhibitors , Growth Inhibitors/chemistry , Growth Inhibitors/pharmacology , Neuregulin-1 , Pyrimidines/pharmacology , Carrier Proteins/chemistry , Carrier Proteins/pharmacology , Glycoproteins/chemistry , Glycoproteins/pharmacology , Humans , Kinetics , Molecular Structure , Peptide Fragments , Phosphorylation/drug effects , Tumor Cells, Cultured
9.
J Med Chem ; 40(12): 1820-6, 1997 Jun 06.
Article in English | MEDLINE | ID: mdl-9191958

ABSTRACT

A series of 6-substituted 4-anilinopyrimido[5,4-d]pyrimidines has been prepared and shown to be potent inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR). These compounds are structurally related to the pyrido[3,2-d]- and pyrido[3,4-d]-pyrimidines previously shown to be EGFR inhibitors. Their structure-activity relationships (SAR) for inhibition of the isolated enzyme more closely resemble those of the [3,2-d] than the [3,4-d] pyridopyrimidine isomers. This suggests the requirement of an aza atom in the 7- but not the 5-position (i.e., a carbon atom in the 5-position) for the enhanced potency shown by 6-N-methylated derivatives in each series. X-ray crystal structures were determined for the three NHMe derivatives 2, 3, and 5c in the pyrido[9,2-d]-, pyrido[3,4-d]-, and pyrimido[5,4-d]-pyrimidine series, respectively. These show that a carbon rather than a nitrogen atom at the 5-position leads to significant conformational changes in the molecule (a longer C5a-C4 bond and a 30 degrees out-of-plane rotation of the phenyl group), due to the requirement to relieve nonbonding interactions between the C5 and N9 protons. Pyrimido[5,4-d]pyrimidine analogues bearing bulky, weakly basic solubilizing side chains linked to the 6-position through a secondary amine generally retained potency both against the isolated enzyme and for inhibition of autophosphorylation of EGFR in intact A431 cells. This agrees with a recent binding model that suggests this general class of compounds binds to EGFR with the 6-position located in an area of comparative bulk tolerance at the entrance to the ATP-binding pocket. While these solubilized pyrimido[5,4-d]pyrimidine analogues were less potent than the NHMe derivative 5c in the isolated enzyme assay, some were considerably superior to 5c (and among the most potent ever reported) as inhibitors of EGFR autophosphorylation in cellular assays.


Subject(s)
Enzyme Inhibitors/chemical synthesis , ErbB Receptors/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/chemical synthesis , Adenosine Triphosphate/metabolism , Binding Sites , Crystallography, X-Ray , Enzyme Inhibitors/pharmacology , ErbB Receptors/metabolism , Humans , Models, Molecular , Molecular Structure , Phosphorylation , Pyrimidines/metabolism , Pyrimidines/pharmacology , Structure-Activity Relationship , Tumor Cells, Cultured
10.
Clin Exp Pharmacol Physiol ; 23(5): 424-7, 1996 May.
Article in English | MEDLINE | ID: mdl-8713682

ABSTRACT

1. Structure-activity relationships are described for the inhibition of the tyrosine kinase activity (phosphorylation of a fragment of phospholipase Cg1) of the epidermal growth factor receptor (EGFR) by 4-anilinoquinazolines. These compounds are competitive inhibitors at the ATP binding site. 2. The preferred side chain is anilino-, substituted at the 3-position with small lipophilic groups. The quinazoline moiety is absolutely required for activity, but substituents on the quinazoline greatly modulate potency, with electron-donating groups favoured. The most potent analogue, the 6,7-dimethoxy derivative (compound 20), has an IC50 of 29 pmol/L and a very high selectivity for the EGFR over other tyrosine kinase enzymes. 3. The present study shows that it is possible to identify small molecules that are very potent, yet highly selective, inhibitors of a single component of the growth signal transduction pathway in cells.


Subject(s)
Adenosine Triphosphate/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Aniline Compounds/pharmacology , ErbB Receptors/drug effects , Quinazolines/pharmacology , Enzyme Inhibitors/pharmacology , In Vitro Techniques , Protein Binding/drug effects , Protein-Tyrosine Kinases/antagonists & inhibitors , Signal Transduction/drug effects , Structure-Activity Relationship
11.
J Med Chem ; 39(9): 1823-35, 1996 Apr 26.
Article in English | MEDLINE | ID: mdl-8627606

ABSTRACT

Following the discovery of the very high inhibitory ability of the 4-[(3-bromophenyl)amino]-quinazolines against the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) (e.g., 3, IC50 0.029 nM), four series of related pyrido[d]pyrimidines bearing electron-donating groups at the 6- or 7-positions have been synthesized and evaluated. The compounds were prepared by nucleophilic substitution of the corresponding 6- and 7-fluoro analogues. While members of all series showed potent inhibitory activity against isolated EGFR, there were important differences between the different isomeric pyrido[d]pyrimidines and the parent quinazolines. Overall, the [3,4-d] and [4,3-d] series were the most potent, followed by the [3,2-d] compounds, with the [2,3-d] analogues being least active. Whereas in the parent quinazoline series the addition of steric bulk to a 6- or 7-NH2 substituent (i.e., NHMe and NMe2 groups) dramatically decreased potency, no such trend was discernable in the [3,2-d] series. Furthermore, in the 7-substituted pyrido[4,3-d]- and 6-substituted pyrido[3,4-d]pyrimidine series, and to a limited extent in the 7-substituted pyrido[2,3-d] series, such substitution increased potency dramatically, to the extent that the 7-(methylamino)pyrido[4,3-d]pyrimidine (5f) (IC50 0.13 nM) and 6-(methylamino)pyrido[3,4-d]pyrimidine (7f) (IC50 0.008 nM) constitute important new leads. Selected compounds were evaluated for their ability to inhibit EGFR autophosphorylation in A431 cells, and a positive quantitative correlation was found between this activity and inhibitory activity against the isolated enzyme.


Subject(s)
ErbB Receptors/antagonists & inhibitors , Pyrimidines/pharmacology , ErbB Receptors/metabolism , Humans , Isomerism , Magnetic Resonance Spectroscopy , Phosphorylation , Pyrimidines/chemistry , Structure-Activity Relationship , Tumor Cells, Cultured
12.
J Med Chem ; 39(4): 918-28, 1996 Feb 16.
Article in English | MEDLINE | ID: mdl-8632415

ABSTRACT

Following the discovery of 4-[(3-bromophenyl)amino]-6,7-dimethoxyquinazoline (4; PD 153035) as an extremely potent (IC(50) 0.025 nM) inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR), several fused tricyclic quinazoline analogues have been prepared and evaluated for their ability to inhibit the enzyme. The most potent compound was the linear imidazo[4,5-g]quinazoline (8), which exhibited an IC(50) of 0.008 nM for inhibition of phosphorylation of a fragment of phospholipase C-gamma-1 as substrate. While N-methyl analogues of 8 showed similar potency, analogous N-[2-(dimethylamino)ethyl] derivatives were less effective. The next most potent compounds were the linear pyrazoloquinazolines (19 and 20) (IC(50)s 0.34 and 0.44 nM) and pyrroloquinazoline (21) (IC(50) 0.44nM), while several other linear tricyclic ring systems of similar geometry to 8 (triazolo-, thiazolo-, and pyrazinoquinazolines) were less effective. In the imidazo[4,5-g]quinazoline and pyrroloquinazoline series, the corresponding angular isomers were also much less effective than the linear ones. These results are consistent with structure-activity relationship studies previously developed for the 4-[(3-bromophenyl)amino] quinazolines, which suggested that small electron-donating substituents at the 6- and 7-positions were desirable for high potency. Cellular studies of the linear imidazoloquinazoline 8 show that it can enter cells and rapidly and very selectively shut down EGF-stimulated signal transmission by binding competitively at the ATP site of the EGFR.


Subject(s)
Enzyme Inhibitors/chemical synthesis , ErbB Receptors/antagonists & inhibitors , Growth Substances/pharmacology , Heterocyclic Compounds/chemical synthesis , Imidazoles/chemical synthesis , Protein-Tyrosine Kinases/antagonists & inhibitors , Quinazolines/chemical synthesis , 3T3 Cells , Adenosine Triphosphate/metabolism , Animals , Binding Sites , Carcinoma, Squamous Cell , Cell Cycle/drug effects , Cell Line , DNA/biosynthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Epidermal Growth Factor/pharmacology , Fibroblast Growth Factor 2/pharmacology , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/pharmacology , Humans , Imidazoles/chemistry , Imidazoles/pharmacology , Indicators and Reagents , Magnetic Resonance Spectroscopy , Mice , Mitosis/drug effects , Molecular Structure , Phosphorylation , Platelet-Derived Growth Factor/pharmacology , Protein-Tyrosine Kinases/chemistry , Quinazolines/chemistry , Quinazolines/pharmacology , Structure-Activity Relationship , Thymidine/metabolism
13.
J Med Chem ; 39(1): 267-76, 1996 Jan 05.
Article in English | MEDLINE | ID: mdl-8568816

ABSTRACT

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.


Subject(s)
Aniline Compounds/pharmacology , ErbB Receptors/antagonists & inhibitors , Quinazolines/pharmacology , Aniline Compounds/chemical synthesis , Aniline Compounds/chemistry , Binding Sites , Blotting, Western , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Humans , Kinetics , Molecular Conformation , Molecular Structure , Phosphorylation/drug effects , Protein Binding , Quinazolines/chemical synthesis , Quinazolines/chemistry , Structure-Activity Relationship , Tumor Cells, Cultured
14.
J Med Chem ; 38(18): 3482-7, 1995 Sep 01.
Article in English | MEDLINE | ID: mdl-7658435

ABSTRACT

A series of 4-substituted quinazolines and related compounds have been prepared and evaluated for their ability to inhibit the tyrosine kinase activity of the epidermal growth factor receptor on a phospholipase C-gamma 1-derived substrate. The results show a narrow structure-activity relationship (SAR) for the basic ring system, with quinazoline being the preferred chromophore and benzylamino and anilino the preferred side chains. In the 4-anilino series, substitution on the 3-position of the phenyl ring with small lipophilic electron-withdrawing groups provided analogues with enhanced potency. Two series of compounds [4-(phenylmethyl)amino and 4-(3-bromophenyl)amino] were studied to determine SARs for quinazoline substituents. In the more active 4-(3-bromophenyl)amino series, electron-donating groups (NH2, OMe) at the 6- or 7-position increased activity, in a pattern consistent with a requirement for high electron density in the vicinity of the 8-position of the quinazoline ring. The 6,7-dimethoxy derivatives were the most effective in both series, with the 4-(3-bromophenyl)amino derivative (3) having an IC50 of 0.029 nM, making it by far the most potent reported inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor enzyme.


Subject(s)
ErbB Receptors/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Quinazolines/pharmacology , Adenosine Triphosphate/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Amino Acid Sequence , ErbB Receptors/metabolism , Humans , Molecular Sequence Data , Quinazolines/chemistry , Structure-Activity Relationship , Tumor Cells, Cultured , Type C Phospholipases/metabolism
15.
Drug Metab Dispos ; 23(3): 363-8, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7628302

ABSTRACT

5,6-Dimethylxanthenone-4-acetic acid (DXAA) is a synthetic xanthenone derivative that is active against murine solid tumors and is being formulated for clinical trials. This study used the isolated perfused rat liver to compare the hepatic metabolism and biliary excretion of DXAA with flavone-8-acetic acid (FAA), a synthetic flavonoid undergoing clinical evaluation as an anticancer drug. Perfusate, bile, and liver samples were assayed for parent drug and metabolites by HPLC. Three FAA metabolites were present in bile, and one of these coeluted with FAA acyl glucuronide. Alkaline hydrolysis of high-dose DXAA bile samples resulted in the disappearance of 5 of 7 metabolite peaks. One biliary metabolite was identified by mass spectrometry as the acyl glucuronide and its presence in bile accounted for > 50% of the DXAA dose. A second compound that was resistant to alkaline hydrolysis was characterized as a hydroxylated DXAA metabolite. A total of 28% of the high dose DXAA was recovered unchanged in the perfusate, liver, and bile, compared with 11% of the low dose DXAA and 40% of the FAA dose. Protein binding of DXAA in perfusate was saturable, ranging from 94.5% at 112 microM to 72.4% at 1125 microM, whereas binding in human plasma was > 99% at concentrations between 11.5 and 1243 microM. This study demonstrates that DXAA undergoes extensive acyl glucuronidation followed by biliary excretion in the isolated perfused rat liver. Its hepatic metabolism may be saturable, and DXAA seems to be more extensively metabolized than FAA. Finally, DXAA protein binding in human plasma is high and not dose-dependent at concentrations likely to be clinically relevant.


Subject(s)
Antineoplastic Agents/metabolism , Flavonoids/metabolism , Glucuronates/chemistry , Liver/metabolism , Xanthenes/metabolism , Xanthones , Animals , Bile/metabolism , Flavonoids/chemistry , Glucuronic Acid , In Vitro Techniques , Male , Perfusion , Protein Binding , Rats , Rats, Sprague-Dawley
16.
J Med Chem ; 38(1): 58-67, 1995 Jan 06.
Article in English | MEDLINE | ID: mdl-7837241

ABSTRACT

A series of 3-substituted 2,2'-dithiobis(1H-indoles) were synthesized 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, to extend the available structure-activity relationships for this series. The majority of the compounds were prepared either by reaction of 2-chloro-1-methylindole-3-carbonyl chloride with amines, followed by thiomethylation, demethylation, and oxidative dimerization, or by reaction of isocyanates with the anion of 1-methyl-2-indolinethione followed by dimerization. Overall, inhibitory activity is retained by analogues having a wide variety of side chains. A series of 3-carboxamide analogues had moderate to good activity against isolated EGFR (IC50s 1-20 microM), with monoalkyl substitution of the carboxamide being optimal. Polar side chains were generally less effective than lipophilic ones, with benzyl being particularly effective. However, N,N-disubstitution was the most effective pattern for inhibition of pp60v-src. A variety of substituted N-phenylcarboxamides had lower activity against EGFR than the parent derivative, and a N-thienylcarboxamide also had low activity. A series of 3-ketones, including methyl, phenyl, and furyl derivatives, showed moderate activity against the pp60v-src kinase, but were less effective against EGFR. The mechanism of inhibition of both kinases by these drugs was shown to be noncompetitive with respect to both ATP and peptide substrate. Selected compounds inhibited the growth of Swiss 3T3 cells with IC50s in the low micromolar range and inhibited bFGF-mediated intracellular tyrosine phosphorylation in the same cell line. Thiol inhibits the effects of the compounds, suggesting that one possible mechanism of inhibition is thiol-disulfide exchange with thiol-containing residues in the catalytic sites. Crystal structures of two representative compounds show a folded, V-shaped structure, with the disulfide bridge exposed, consistent with this hypothesis.


Subject(s)
ErbB Receptors/antagonists & inhibitors , Indoles/chemical synthesis , Indoles/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , 3T3 Cells/drug effects , Animals , Cell Division/drug effects , Chemical Phenomena , Chemistry, Physical , Crystallography, X-Ray , Indoles/chemistry , Ketones/chemical synthesis , Ketones/chemistry , Ketones/pharmacology , Kinetics , Mice , Molecular Structure , Structure-Activity Relationship
17.
Xenobiotica ; 24(7): 635-47, 1994 Jul.
Article in English | MEDLINE | ID: mdl-7975728

ABSTRACT

1. Xanthenone-4-acetic acid (XAA) is an experimental antitumour agent which resembles flavone-8-acetic acid in its induction of cytokine synthesis, nitric oxide production and tumour haemorrhagic necrosis. We have investigated the excretion and metabolic fate of XAA in the BDF1 mouse. 2. XAA was administered intravenously at the maximal tolerated dose (1090 mumol/kg). Urine, plasma and bile were collected and subjected to analysis by hplc. Urine samples demonstrated labile metabolites which released XAA following incubation with beta-glucuronidase/sulphatase or at pH 9.0. The structures of isolated XAA metabolites were characterized by ms or 1H-NMR spectra at 400 MHz. 3. The major metabolite pathway of XAA involves conjugation with glucuronic acid, since the resulting metabolite, XAA acyl glucuronide, accounts for 25% of the dose excreted in the urine. Other metabolite pathways include alpha-oxidation of the acetic acid side chain and aromatic hydroxylation of the xanthenone ring.


Subject(s)
Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacokinetics , Xanthenes/metabolism , Xanthenes/pharmacokinetics , Animals , Bile/metabolism , Biliary Tract/metabolism , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Glucuronidase/metabolism , Hydrolysis , Male , Mice , Mice, Inbred Strains , Sulfatases/metabolism , Xanthenes/urine
18.
J Med Chem ; 37(13): 2033-42, 1994 Jun 24.
Article in English | MEDLINE | ID: mdl-8027985

ABSTRACT

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.


Subject(s)
Indoles/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , 3T3 Cells , Animals , Blotting, Western , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , ErbB Receptors/metabolism , Fibroblast Growth Factor 2/metabolism , Humans , Indoles/chemical synthesis , Indoles/chemistry , Magnetic Resonance Spectroscopy , Mice , Oncogene Protein pp60(v-src)/antagonists & inhibitors , Oxidation-Reduction , Phosphorylation/drug effects , Structure-Activity Relationship , Tumor Cells, Cultured
19.
J Med Chem ; 36(17): 2459-69, 1993 Aug 20.
Article in English | MEDLINE | ID: mdl-8355247

ABSTRACT

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.


Subject(s)
ErbB Receptors/drug effects , Indoles/chemical synthesis , Protein-Tyrosine Kinases/antagonists & inhibitors , Thiones/chemical synthesis , Animals , Cells, Cultured , Fibroblasts/drug effects , Humans , Indoles/chemistry , Indoles/pharmacology , Mice , Structure-Activity Relationship , Thiones/chemistry , Thiones/pharmacology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/enzymology
20.
Anticancer Drug Des ; 7(5): 403-14, 1992 Oct.
Article in English | MEDLINE | ID: mdl-1388631

ABSTRACT

A series of 9-oxo-9,10-dihydroacridine-4-acetic acids (acridone-4-acetic acids) were prepared by Jourdan-Ullmann condensation of 2-halobenzoic acids with 2-aminophenylacetic acids, followed by H2SO4-induced cyclodehydration of the resulting 2-[2-(carboxymethyl)phenylamino]benzoic acids. These were evaluated for their ability to induce haemorrhagic necrosis in transplanted colon 38 tumours in mice, using a short-term histology assay. The results broadly paralleled those seen previously for xanthenone-4-acetic acids, with 1-, 2- and 7-substitution being dystherapeutic, and substitution at the 5- and 6-positions by lipophilic groups increasing activity. While some analogues were as active as xanthenone-4-acetic acids in the histology assay and gave significant growth delays against colon 38 tumours in vivo, as a class the 9-oxo-9,10-dihydroacridine-4-acetic acids were generally less potent than the xanthenone-4-acetic acids.


Subject(s)
Colonic Neoplasms/pathology , Drug Design , Xanthenes/chemistry , Xanthenes/pharmacology , Animals , Colonic Neoplasms/drug therapy , Mice , Necrosis , Structure-Activity Relationship
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