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Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-ß-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential ß-lactamase stable ß-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.

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Discovery of sofosbuvir has radically changed hepatitis C treatment and nucleoside/tide NS5B inhibitors are now viewed as one of the key components in combination therapies with other direct-acting antiviral agents. As part of our program to identify new nucleoside inhibitors of HCV replication, we now wish to report on the discovery of ß-d-2'-deoxy-2'-dichlorouridine nucleotide prodrugs as potent inhibitors of HCV replication. Although, cytidine analogues have long been recognized to be metabolized to both cytidine and uridine triphosphates through the action of cytidine deaminase, uridine analogues are generally believed to produce exclusively uridine triphosphate. Detailed investigation of the intracellular metabolism of our newly discovered uridine prodrugs, as well as of sofosbuvir, has now revealed the formation of both uridine and cytidine triphosphates. This occurs, not only in vitro in cell lines, but also in vivo upon oral dosing to dogs.

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A set of low molecular weight compounds containing a hydroxyethylamine (HEA) core structure with different prime side alkyl substituted 4,5,6,7-tetrahydrobenzazoles and one 4,5,6,7-tetrahydropyridinoazole was synthesized. Striking differences were observed on potencies in the BACE-1 enzymatic and cellular assays depending on the nature of the heteroatoms in the bicyclic ring, from the low active compound 4 to inhibitor 6, displaying BACE-1 IC(50) values of 44 nM (enzyme assay) and 65 nM (cell-based assay).

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Two types of P1-P3-linked macrocyclic renin inhibitors containing the hydroxyethylene isostere (HE) scaffold just outside the macrocyclic ring have been synthesized. An aromatic or aliphatic substituent (P3sp) was introduced in the macrocyclic ring aiming at the S3 subpocket (S3sp) in order to optimize the potency. A 5-6-fold improvement in both the K(i) and the human plasma renin activity (HPRA)IC(50) was observed when moving from the starting linear peptidomimetic compound 1 to the most potent macrocycle 42 (K(i) = 3.3 nM and HPRA IC(50) = 7 nM). Truncation of the prime side of 42 led to 8-10-fold loss of inhibitory activity in macrocycle 43 (K(i) = 34 nM and HPRA IC(50) = 56 nM). All macrocycles were epimeric mixtures in regard to the P3sp substituent and X-ray crystallographic data of the representative renin macrocycle 43 complex showed that only the S-isomer buried the substituent into the S3sp. Inhibitory selectivity over cathepsin D (Cat-D) and BACE-1 was also investigated for all the macrocycles and showed that truncation of the prime side increased selectivity of inhibition in favor of renin.

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Novel NS3/4A protease inhibitors comprising quinazoline derivatives as P2 substituent were synthesized. High potency inhibitors displaying advantageous PK properties have been obtained through the optimization of quinazoline P2 substituents in three series exhibiting macrocyclic P2 cyclopentane dicarboxylic acid and P2 proline urea motifs. For the quinazoline moiety it was found that 8-methyl substitution in the P2 cyclopentane dicarboxylic acid series improved on the metabolic stability in human liver microsomes. By comparison, the proline urea series displayed advantageous Caco-2 permeability over the cyclopentane series. Pharmacokinetic properties in vivo were assessed in rat on selected compounds, where excellent exposure and liver-to-plasma ratios were demonstrated for a member of the 14-membered quinazoline substituted P2 proline urea series.

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A novel series of P3-truncated macrocyclic HCV NS3/4A protease inhibitors containing a P2 proline-urea or carbamate scaffold was synthesized. Very potent inhibitors were obtained through the optimization of the macrocycle size, urea and proline substitution, and bioisosteric replacement of the P1 carboxylic acid moiety. Variation of the lipophilicity by introduction of small lipophilic substituents resulted in improved PK profiles, ultimately leading to compound 13Bh, an extremely potent (K(i)=0.1 nM, EC(50)=4.5 nM) and selective (CC(50) (Huh-7 cells)>50 microM) inhibitor, displaying an excellent PK profile in rats characterized by an oral bioavailability of 54% and a high liver exposure after oral administration.

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We have identified 1H-benzylindole analogues as a novel series of human immunodeficiency virus (HIV) integrase inhibitors with antiretroviral activities against different strains of HIV type 1 (HIV-1), HIV-2, and simian immunodeficiency virus strain MAC(251) [SIV(MAC(251))]. Molecular modeling and structure-activity relationship-based optimization resulted in the identification of CHI/1043 as the most potent congener. CHI/1043 inhibited the replication of HIV-1(III(B)) in MT-4 cells at a 50% effective concentration (EC(50)) of 0.60 microM, 70-fold below its cytotoxic concentration. Equal activities against HIV-1(NL4.3), HIV-2(ROD), HIV-2(EHO), and SIV(MAC(251)) were observed. CHI/1043 was equally active against virus strains resistant against inhibitors of reverse transcriptase or protease. Replication of both X4 and R5 strains in peripheral blood mononuclear cells was sensitive to the inhibitory effect of CHI/1043 (EC(50), 0.30 to 0.38 microM). CHI/1043 inhibited integrase strand transfer activity in oligonucleotide-based enzymatic assays at low micromolar concentrations. Time-of-addition experiments confirmed CHI/1043 to interfere with the viral replication cycle at the time of retroviral integration. Quantitative Alu PCR corroborated that the anti-HIV activity is based upon the inhibition of proviral DNA integration. An HIV-1 strain selected for 70 passages in the presence of CHI/1043 was evaluated genotypically and phenotypically. The mutations T66I and Q146K were present in integrase. Cross-resistance to other integrase strand transfer inhibitors, such as L-708,906, the naphthyridine analogue L-870,810, and the clinical drugs GS/9137 and MK-0518, was observed. In adsorption, distribution, metabolism, excretion, and toxicity studies, antiviral activity was strongly reduced by protein binding, and metabolization in human liver microsomes was observed. Transport studies with Caco cells suggest a low oral bioavailability.

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