Inhibitors of NF-kappaB and AP-1 gene expression: SAR studies on the pyrimidine portion of 2-chloro-4-trifluoromethylpyrimidine-5-[N-(3', 5'-bis(trifluoromethyl)phenyl)carboxamide].

We investigated the structure-activity relationship studies of N-[3, 5-bis(trifluoromethyl)phenyl][2-chloro-4-(trifluoromethyl)pyrimidin-5 -yl]carboxamide (1), an inhibitor of transcription mediated by both NF-kappaB and AP-1 transcription factors, with the goal of improving its potential oral bioavailability. Compounds were examined for cell-based activity, were fit to Lipinski's rule of 5, and were examined for potential gastrointestinal permeability using the intestinal epithelial cell line, Caco-2. Selected groups were substituted at the 2-, 4-, and 5-positions of the pyrimidine ring using solution-phase combinatorial methodology. The introduction of a fluorine in the place of 2-chlorine of 1 resulted in a compound with comparable activity. However, other substitutions at the 2-position resulted in a loss of activity. The trifluoromethyl group at the 4-position could be replaced with a methyl, ethyl, chlorine, or phenyl without a substantial loss of activity. The carboxamide group at the 5-position is critical for activity. If it was moved to the 6-position, the activity was lost. The 2-methyl analogue of 1 (81) showed comparable in vitro activity and improved Caco-2 permeability compared to 1.

Novel inhibitors of AP-1 and NF-kappaB mediated gene expression: structure-activity relationship studies of ethyl 4-[(3-methyl-2,5-dioxo(3-pyrrolinyl))amino]-2-(trifluoromethyl)++ +pyrimidi ne-5-carboxylate.

In an effort to identify novel inhibitors of AP-1 and NF-kappaB mediated transcriptional activation, several analogues of ethyl 4-[(3-methyl-2,5-dioxo(3-pyrrolinyl))amino]-2-(trifluoromethyl)pyr imidine-5-carboxylate (1) were synthesized and tested in two in vitro assays. The 2-(2'-thienyl) substituted compound (11) was identified as the most potent in this series.

2-Chloro-4-(trifluoromethyl)pyrimidine-5-N-(3',5'- bis(trifluoromethyl)phenyl)-carboxamide: a potent inhibitor of NF-kappa B- and AP-1-mediated gene expression identified using solution-phase combinatorial chemistry.

Described is the identification of a novel series of compounds that blocks the activation of two key transcription factors, AP-1 and NF-kappa B. These transcription factors regulate the expression of several critical proinflammatory proteins and cytokines and represent attractive targets for drug discovery. Through the use of high throughput screening and solution-phase parallel synthesis, inhibitors of both NF-kappa B and AP-1 were identified. In subsequent testing, these compounds were also shown to block both IL-2 and IL-8 levels in the same cells. One of the most potent compounds in this series, 28, was active in several animal models of inflammation and immunosuppression, thus validating the importance of AP-1 and NF-kappa B as potential therapeutic targets. The synthesis and preliminary structure-activity relationships of these compounds is addressed.

Immunospecific reduction of antioligonucleotide antibody-forming cells with a tetrakis-oligonucleotide conjugate (LJP 394), a therapeutic candidate for the treatment of lupus nephritis.

A discrete tetravalent conjugate, 7a (LJP 394), consisting of four oligonucleotides attached to a common carrier or platform was prepared. Single-stranded oligonucleotide 20-mers consisting of alternating deoxycytidine-deoxyadenosine nucleotides, (CA)10, were attached to a tetrabromoacetylated platform by displacement with sulfhydryl-terminated linkers. The tetrabromoacetylated platform 3a was synthesized in three steps using triethylene glycol bis-(chloroformate). The single-stranded conjugate was characterized by polyacrylamide gel electrophoresis, DNA sequencing, phosphate analysis, carbon and nitrogen combustion analysis, and correlation of stoichiometry to conversion in the conjugation process. HPLC and capillary electrophoretic methods were developed to evaluate purity. The tetrakis, single-stranded conjugate was annealed with a stoichiometric amount of a complementary single-stranded oligonucleotide 20-mer consisting of alternating thymidine-deoxyguanosine nucleotides, (TG)10. The double-stranded conjugate LJP 394 was characterized by melt temperature and hyperchromicity, phosphate analysis, and carbon and nitrogen combustion analysis. LJP 394 inhibits binding of DNA to anti-double-stranded oligonucleotide antibodies and reduces anti-oligonucleotide-specific plaque (antibody)-forming cells in an immunized mouse model by a proposed mechanism involving cross-linking B cell surface immunoglobins.

Evaluation of functional analogs of CC-1065 and the duocarmycins incorporating the cross-linking 9a-chloromethyl-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-on e (C2BI) alkylation subunit.

The DNA alkylation properties and in vitro cytotoxic activity of a series of analogs of CC-1065 and the duocarmycins incorporating the 9a-chloromethyl-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (C2BI) alkylation subunit are detailed. The C2BI-based agents have been shown to alkylate DNA within the minor groove in a fashion analogous to CC-1065 or duocarmycin. The stereoelectronically-controlled adenine N3 addition to the least substituted cyclopropane carbon occurs with a selectivity that represents a composite of the two enantiomers of the corresponding CBI-based agents. Additional high affinity alkylation sites were detected which were not prominent alkylation sites for either enantiomer of the CBI-based agents. Such sites may represent induced high affinity alkylation sites resulting from DNA cross-linking following complementary strand alkylation at a high affinity alkylation site and each such site detected proved consistent with predicted models of an adenine-adenine cross-linking event. Further, consistent with this interpretation, the C2BI agents were shown to constitute efficient cross-linking agents with DNA cross-linking being observed at the same concentrations as DNA alkylation. In comparison to the parent CBI-based agents, the C2BI-based agents proved to be approximately 100-10,000x less effective at DNA alkylation and 100-10,000x less potent in cytotoxic assays. This is suggested to be the consequence of a significant steric deceleration of the adenine N3 alkylation reaction attributable to the additional 9a-chloromethyl substituent. Consistent with this interpretation, the noncovalent binding constant of C2BI-CDPI2 for poly[dA]-poly[dA]-poly[dT] proved nearly identical to that of CDPI3 under kinetic binding conditions, and prolonged incubation of C2BI-CDPI2 with poly[dA]-poly[dT] (72 h, 25 degrees C) provided covalent complexes with a helix stabilization comparable to that observed with (+)- or (-)-CPI-CDPI2 indicating that the size of the C2BI subunit inhibits but does not preclude productive DNA alkylation.

Structures of two conformationally defined phenylethanolamines: exo-1,4-epoxy-2-formamido-1,2,3,4-tetrahydro-8-trifluoromethylnaphthale ne and exo-1,4-epoxy-2-formamido-1,2,3,4-tetrahydro-6-trifluoromethylnaphthale ne.

(+/-)-exo-1,4-Epoxy-2-formamido-1,2,3,4-tetrahydro-8- trifluoromethylnaphthalene (1), C12H10F3NO2, Mr = 257.21, Pccn, a = 8.895 (1), b = 19.968 (5), c = 12.656 (3) A, V = 2247.9 (8) A3, Z = 8, Dx = 1.520 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 1.49 cm-1, F(000) = 1056, T = 297 K, R = 0.0466 for 1481 independent reflections collected. The torsion angle for N(10)-C(2)-C(1)-C(8a) is 167.4 (2) degrees. (+/-)-exo-1,4-Epoxy-2-formamido-1,2,3,4-tetrahydro-6- trifluoromethylnaphthalene, (2), C12H10F3NO2, Mr = 257.21, P2(1)2(1)2(1), a = 8.52 (2), b = 26.15 (2), c = 5.06 (5) A, V = 1127 (11) A3, Z = 4, Dx = 1.516 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 1.29 cm-1, F(000) = 528, T = 297 K, R = 0.108 for 932 independent reflections collected. The torsion angle N(10)-C(2)-C(1)-C(8a) is 172.5 (6) degrees; the formamido group in both (1) and (2) is exo. X-ray studies on (1) suggest a hydrogen bond between N(10) and O(12) and similarly for (2).