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Structure-activity relationships for inhibition of erbB1, erbB2, and erbB4 were determined for a series of quinazoline- and pyrido[3,4-d]pyrimidine-based analogues of the irreversible pan-erbB inhibitor, canertinib. Cyclic amine bearing crotonamides were determined to provide rapid inhibition of cellular erbB1 autophosphorylation and good metabolic stability in liver microsome and hepatocyte assays. The influence of 4-anilino substitution on pan-erbB inhibitory potency was investigated. Several anilines were identified as providing potent, reversible pan-erbB inhibition. Optimum 4- and 6-substituents with known 7-substituents provided preferred irreversible inhibitors for pharmacodynamic testing in vivo. Quinazoline 54 and pyrido[3,4-d]pyrimidine 71 were identified as clearly superior to canertinib. Both compounds possess a piperidinyl crotonamide Michael acceptor and a 3-chloro-4-fluoroaniline, indicating these as optimized 6- and 4-substituents, respectively. Pharmacokinetic comparison of compounds 54 and 71 across three species selected compound 54 as the preferred candidate. Compound 54 (PF-00299804) has been assigned the nomenclature of dacomitinib and is currently under clinical evaluation.

Structure-activity relationships for inhibition of erbB1, erbB2, and erbB4 were determined for a series of alkynamide analogues of quinazoline- and pyrido[3,4-d]pyrimidine-based compounds. The compounds were prepared by coupling the appropriate 6-aminoquinazolines or 6-aminopyrido[3,4-d]pyrimidines with alkynoic acids, using EDCI.HCl in pyridine. The compounds showed pan-erbB enzyme inhibition but were on average about 10-fold more potent against erbB1 than against erbB2 and erbB4. For cellular inhibition, the nature of the alkylating side chains was an important determinant, with 5-dialkylamino-2-pentynamide type Michael acceptors providing the highest potency. This is suggested to be due to an improved ability of the amine to participate in an autocatalysis of the Michael reaction with enzyme cysteine residues. Pyrido[3,4-d]pyrimidine analogue 39 was selected for in vivo evaluation and achieved tumor regressions at 10 mg/kg in the A431 human epidermoid carcinoma and at 40 mg/kg for the SF767 human glioblastoma and the SKOV3 human ovarian carcinoma. Complete stasis was observed at 40 mg/kg in the BXPC3 human pancreatic carcinoma as well as in the H125 human non-small-cell lung carcinoma.

4-Anilinoquinazoline- and 4-anilinopyrido[3,4-d]pyrimidine-6-acrylamides are potent pan-erbB tyrosine kinase inactivators, and one example (CI-1033) is in clinical trial. A series of analogues with a variety of Michael acceptor units at the 6-position were prepared to define the structural requirements for irreversible inhibition. A particular goal was to determine whether additional functions to increase solubility could be appended to the Michael acceptor. Substituted acrylamides were prepared by direct acylation of the corresponding 6-amines with the requisite acid or acid chloride. Vinylsulfonamide derivatives were obtained by acylation of the amines with chloroethylsulfonyl chloride followed by base-promoted elimination. Vinylsulfone and vinylsulfine derivatives were prepared by oxidation and base elimination of a hydroxyethylthio intermediate. 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. Substitution at the nitrogen of the acrylamide was tolerated only with a methyl group; larger substituents were dystherapeutic, and no substitution at all was tolerated at the acrylamide alpha-carbon. In contrast, while electron-donating groups at the acrylamide beta-carbon were not useful, even quite large electron-withdrawing groups (which increase its electrophilicity) were tolerated. A series of derivatives with solubility-enhancing substituents linked to the acrylamide beta-carbon via amides were potent irreversible inhibitors of isolated EGFR (IC50s = 0.4-1.1 nM), with weakly basic morpholine and imidazole derivatives being the best. Vinylsulfonamides were also potent and irreversible inhibitors, but vinylsulfones and vinylsulfines were reversible and only poorly active. Two compounds were evaluated against A431, H125, and MCF-7 xenografts in nude mice but were inferior in these assays to the clinical trial compound CI-1033.

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.

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.

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.

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.

Several elaborations of the fundamental anilinopyrimidine pharmacophore have been reported as potent and selective inhibitors of the epidermal growth factor receptor (EGFr) tyrosine kinase. This paper reports on a series of inhibitors whereby some 6,5-bicyclic heteroaromatic systems were fused through their C-2 and C-3 positions to this anilinopyrimidine pharmacophore. Although the resulting tricycles did not produce the enormous potency of some of the (5/6),6,6-bicyclic systems, the best of them had IC(50)s for the EGFr TK around 1 nM. Investigation of 4-position side chains in the indolopyrimidines confirmed that m-bromoaniline was an optimal substituent for potency. Investigation of substitution within the C-(benzo)ring of benzothienopyrimidines confirmed that introduction of an extra ring can change sharply the effects of substituents when compared to similar bicyclic nuclei, and only two substituents were found which even moderately enhanced inhibitory activity over the parent compound for this series.

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.

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].

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.

A series of 5-amino- and 5-hydroxyquinolone antibacterials substituted at C7 with a select group of common piperazinyl and 3-aminopyrrolidinyl side chains was prepared. These 5-substituted derivatives were compared to the analogous 5-hydrogen compounds for antiinfective activity by using DNA gyrase inhibition, minimum inhibitory concentrations against a variety of bacteria, and in vivo efficacy in the mouse infection model. The influence on the structure-activity relationships of varied substituents at C8 (H, F, Cl) and N1 (ethyl, cyclopropyl, difluorophenyl) was also studied. The results showed that several of the structure-activity conclusions regarding side-chain bulk at C7, the effect of halogen at C8, and the effect of the C5-amino group were greatly influenced by the choice of the N1-substituent. Several outstanding broad spectrum quinolones were identified in this work. In particular, the spectrum and potency of the 7-piperazinyl quinolones could be greatly enhanced by the judicious choice of C5-, C8-, and N1-substituents.

Synthese de (N 6 -[diaryl-2,2 ethyl]) adenosines, en particulier l'adenosine du titre, a partir de la (chloro-6 ribofuranosyl-9) purine