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Perforin is a key effector protein in the vertebrate immune system and is secreted by cytotoxic T lymphocytes and natural killer cells to help eliminate virus-infected and transformed target cells. The ability to modulate perforin activity in vivo could be extremely useful, especially in the context of bone marrow stem cell transplantation where early rejection of immunologically mismatched grafts is driven by the recipient's natural killer cells, which overwhelmingly use perforin to kill their targets. Bone marrow stem cell transplantation is a potentially curative treatment for both malignant and nonmalignant disorders, but when the body recognizes the graft as foreign, it is rejected by this process, often with fatal consequences. Here we report optimization of a previously identified series of benzenesulfonamide-based perforin inhibitors for their physicochemical and pharmacokinetic properties, resulting in the identification of 16, the first reported small molecule able to prevent rejection of transplanted bone marrow stem cells in vivo by blocking perforin function.

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.

The l-type amino acid transporter 1 (LAT1) is a transmembrane protein carrying bulky and neutral amino acids into cells. LAT1 is overexpressed in several types of tumors, and its inhibition can result in reduced cancer cell growth. However, known LAT1 inhibitors lack selectivity over other transporters. In the present study, we designed and synthesized a novel selective LAT1 inhibitor (1), which inhibited the uptake of LAT1 substrate, l-leucin as well as cell growth. It also significantly potentiated the efficacy of bestatin and cisplatin even at low concentrations (25 μM). Inhibition was slowly reversible, as the inhibitor was able to be detached from the cell surface and blood-brain barrier. Moreover, the inhibitor was metabolically stable and selective toward LAT1. Since the inhibitor was readily accumulated into the prostate after intraperitoneal injection to the healthy mice, this compound may be a promising agent or adjuvant especially for the treatment of prostate cancer.

A series of novel 5-arylidene-2-thioxoimidazolidin-4-ones were investigated as inhibitors of the lymphocyte-expressed pore-forming protein perforin. Structure-activity relationships were explored through variation of an isoindolinone or 3,4-dihydroisoquinolinone subunit on a fixed 2-thioxoimidazolidin-4-one/thiophene core. The ability of the resulting compounds to inhibit the lytic activity of both isolated perforin protein and perforin delivered in situ by natural killer cells was determined. A number of compounds showed excellent activity at concentrations that were nontoxic to the killer cells, and several were a significant improvement on previous classes of inhibitors, being substantially more potent and soluble. Representative examples showed rapid and reversible binding to immobilized mouse perforin at low concentrations (≤2.5 μM) by surface plasmon resonance and prevented formation of perforin pores in target cells despite effective target cell engagement, as determined by calcium influx studies. Mouse PK studies of two analogues showed T1/2 values of 1.1-1.2 h (dose of 5 mg/kg i.v.) and MTDs of 60-80 mg/kg (i.p.).

A new series of MEK1 inhibitors, the 4-anilino-5-carboxamido-2-pyridones, were designed and synthesized using a combination of medicinal chemistry, computational chemistry, and structural elucidation. The effect of variation in the carboxamide side chain, substitution on the pyridone nitrogen, and replacement of the 4'-iodide were all investigated. This study afforded several compounds which were either equipotent or more potent than the clinical candidate CI-1040 (1) in an isolated enzyme assay, as well as murine colon carcinoma (C26) cells, as measured by suppression of phosphorylated ERK substrate. Most notably, pyridone 27 was found to be more potent than 1 in vitro and produced a 100% response rate at a lower dose than 1, when tested for in vivo efficacy in animals bearing C26 tumors.

Heterocyclic phenazinecarboxamides were prepared by condensation of aminoheterocycles and 2-halo-3-nitrobenzoic acids, followed by reductive ring closure and amidation. They showed similar inhibition of paired cell lines that underexpressed topo II or overexpressed P-glycoprotein, indicating a non topo II mechanism of cytotoxicity and indifference to P-glycoprotein mediated multidrug resistance. Compounds with a fused five-membered heterocyclic ring were generally less potent than the pyrido[4,3-a]phenazines. A 4-methoxypyrido[4,3-a]phenazine (IC(50)s 2.5-26 nM) gave modest (ca. 5 day) growth delays in H69/P xenografts with oral dosing.

Bis(9-methylphenazine-1-carboxamides) joined by a variety of dicationic (CH(2))(n)()NR(CH(2))(m)NR(CH(2))(n) linkers of varying length (carboxamide N-N distances from 11.0 to 18.4 A) and rigidity were prepared by reaction of 9-methylphenazine-1-carboxylic acid imidazolide with the appropriate polyamines. The compounds were evaluated for growth inhibitory properties in P388 leukemia, Lewis lung carcinoma, and wild-type (JL(C)) and mutant (JL(A) and JL(D)) forms of human Jurkat leukemia with low levels of topoisomerase II (topo II). The compounds all had IC(50) ratios of1 in the resistant Jurkat lines, consistent with topo II inhibition not being the primary mechanism of action. Analogues joined by an (CH(2))(2)NR(CH(2))(2)NR(CH(2))(2) linker were extremely potent cytotoxins, with selectivity toward the human cell lines, but absolute potencies declined sharply from R = H through R = Me to R = Pr and Bu. In contrast, (CH(2))(2)NR(CH(2))(3)NR(CH(2))(2) compounds showed reverse effects, with the R = Me analogue being more potent than the R = H one as well as being the most potent in the series [IC(50) in JL(C) cells 0.08 nM; superior to that for the clinical bis(naphthalimide) LU 79553]. Overall, the IC(50)s of analogues with linker chains (CH(2))(n)NH(CH(2))(m)NH(CH(2))(n) were inversely proportional to linker length. Constraining the rigidity of the linker chain by incorporating a piperazine ring did not decrease potency significantly. A representative compound bound tightly to DNA with high selectivity for GC sites, compatible with recent work suggesting that compounds of this type place their side chains in the major groove, making specific contacts with guanine bases. Representative compounds were susceptible to transport mediated resistance, being much less effective in cells that overexpressed P-glycoprotein. Overall the results suggest these compounds have a similar mode of action, mediated primarily by poisoning of topo I (possibly with some involvement of topo II). The bis(9-methylphenazine-1-carboxamides) show very high in vitro growth inhibitory potencies compared to their monomeric analogues. Two compounds showed in vivo activity in murine colon 38 syngeneic and HT29 human colon tumor xenograft models using intraperitoneal dosing.

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.

The mixed topoisomerase I/II inhibitor N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) is currently in clinical trial as an anticancer drug. A series of acridine-substituted analogues were prepared, using a new synthetic route to substituted acridine-4-carboxylic acids (conversion of substituted diphenylamine diacid monoesters to the corresponding aldehydes and mild acid-catalyzed ring closure to form the acridines directly). The analogues were evaluated in a panel of cell lines which included wild-type (JLC) and mutant (JLA and JLD) forms of the human Jurkat leukemia line. The latter mutant lines are resistant to topoisomerase II targeted agents due to lower levels of the enzyme. Structure-activity studies suggest that the electronic properties of the substituents do not markedly affect cytotoxicity, but steric bulk is important, with larger groups leading to loss of activity. The compounds fell broadly into two categories. The majority had cytotoxicities similar to (or lower than) that of DACA itself and were equitoxic in all the Jurkat lines, suggesting a relatively greater effect on topoisomerase I compared with topoisomerase II. Most of the 5-substituted derivatives and the 7-Ph compound were more cytotoxic than DACA, but were less effective against JLA and JLD cell lines than in the wild-type JLC, suggesting a mode of cytotoxicity largely mediated by effects on topoisomerase II. Both DACA and selected acridine-substituted analogues were active in the relatively refractory subcutaneous colon 38 tumor model in vivo.

Dihydrofuro[3,4-c]pyridinones are the first class of small molecules reported to inhibit the cytolytic effects of the lymphocyte toxin perforin. A lead structure was identified from a high throughput screen, and a series of analogues were designed and prepared to explore structure-activity relationships around the core bicyclic thioxofuropyridinone and pendant furan ring. This resulted in the identification of a submicromolar inhibitor of the perforin-induced lysis of Jurkat T-lymphoma cells.

A series of acridine-substituted bis(acridine-4-carboxamides) linked by a (CH2)3N(Me)(CH2)3 chain have been prepared by reaction of the isolated imidazolides of the substituted acridine-4-carboxylic acids with N,N-bis(3-aminopropyl)methylamine. These dimeric analogues of the mixed topoisomerase I/II inhibitor N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA), currently in clinical trial, show superior potencies to the corresponding monomeric DACA analogues in a panel of cell lines, including wild-type (JLC) and mutant (JLA and JLD) forms of human Jurkat leukemia. The latter mutant lines are resistant to topoisomerase II targeted agents because of lower levels of the enzyme. Analogues with small substituents (e.g., Me, Cl) at the acridine 5-position were clearly superior, with IC50's as low as 2 nM against the Lewis lung carcinoma and 11 nM against JLC. Larger substituents at any position caused a steady decrease in potency, likely due to lowering of DNA binding affinity. A small series of analogues of the most potent bis(5-methylDACA) compound, with second substituents (Me and Cl) in the 1- or 8- position had broadly similar potencies to the 5-Me compound, indicating that, while the 1- and 8-substituents are acceptable, they add little to the enhancing effect of the 5-methyl group. All of the compounds were at least equitoxic (some up to 4-fold more cytotoxic) against the mutant Jurkat lines than in the wild-type, consistent with a relatively greater effect on topoisomerase I compared with topoisomerase II. The bis(5-methylDACA) compound was found to inhibit the action of purified topoisomerase I in a cell-free assay. Compounds were on average 10-fold less cytotoxic in an MCF7 breast cancer line overexpressing P-glycoprotein than in the wild-type line and showed some selectivity for colon tumor lines in the NCI human tumor cell line panel. Several analogues produced significant growth delays in the relatively refractory subcutaneous colon 38 tumor model in vivo at substantially lower doses than DACA. The bis(acridine-4-carboxamides) represent a new and interesting class of potent topoisomerase inhibitors.