Macrophage Activation Status Rather than Repolarization Is Associated with Enhanced Checkpoint Activity in Combination with PI3Kγ Inhibition.

Suppressive myeloid cells mediate resistance to immune checkpoint blockade. PI3Kγ inhibition can target suppressive macrophages, and enhance efficacy of immune checkpoint inhibitors. However, how PI3Kγ inhibitors function in different tumor microenvironments (TME) to activate specific immune cells is underexplored. The effect of the novel PI3Kγ inhibitor AZD3458 was assessed in preclinical models. AZD3458 enhanced antitumor activity of immune checkpoint inhibitors in 4T1, CT26, and MC38 syngeneic models, increasing CD8+ T-cell activation status. Immune and TME biomarker analysis of MC38 tumors revealed that AZD3458 monotherapy or combination treatment did not repolarize the phenotype of tumor-associated macrophage cells but induced gene signatures associated with LPS and type II INF activation. The activation biomarkers were present across tumor macrophages that appear phenotypically heterogenous. AZD3458 alone or in combination with PD-1-blocking antibodies promoted an increase in antigen-presenting (MHCII+) and cytotoxic (iNOS+)-activated macrophages, as well as dendritic cell activation. AZD3458 reduced IL-10 secretion and signaling in primary human macrophages and murine tumor-associated macrophages, but did not strongly regulate IL-12 as observed in other studies. Therefore, rather than polarizing tumor macrophages, PI3Kγ inhibition with AZD3458 promotes a cytotoxic switch of macrophages into antigen-presenting activated macrophages, resulting in CD8 T-cell-mediated antitumor activity with immune checkpoint inhibitors associated with tumor and peripheral immune activation.

Identification and Optimization of Pyrrolidine Derivatives as Highly Potent Ghrelin Receptor Full Agonists.

Muscle atrophy and cachexia are common comorbidities among patients suffering from cancer, chronic obstructive pulmonary disease, and several other chronic diseases. The peptide hormone ghrelin exerts pleiotropic effects including the stimulation of growth hormone secretion and subsequent increase of insulin-like growth factor-1 levels, an important mediator of muscle growth and repair. Ghrelin also acts on inflammation, appetite, and adipogenesis and therefore has been considered a promising therapeutic target for catabolic conditions. We previously reported on the synthesis and properties of an indane based series of ghrelin receptor full agonists which led to a sustained increase of insulin-like growth factor-1 in a dog pharmacodynamic study. Herein we report on the identification of a series of pyrrolidine or piperidine based full agonists and attempted optimization to give compounds with profiles suitable for progression as clinical candidates.

Discovery of Highly Isoform Selective Orally Bioavailable Phosphoinositide 3-Kinase (PI3K)-γ Inhibitors.

In this paper, we describe the discovery and optimization of a new chemotype of isoform selective PI3Kγ inhibitors. Starting from an HTS hit, potency and physicochemical properties could be improved to give compounds such as 15, which is a potent and remarkably selective PI3Kγ inhibitor with ADME properties suitable for oral administration. Compound 15 was advanced into in vivo studies showing dose-dependent inhibition of LPS-induced airway neutrophilia in rats when administered orally.

Identification and Pharmacological Profile of an Indane Based Series of Ghrelin Receptor Full Agonists.

Cachexia and muscle wasting are very common among patients suffering from cancer, chronic obstructive pulmonary disease, and other chronic diseases. Ghrelin stimulates growth hormone secretion via the ghrelin receptor, which subsequently leads to increase of IGF-1 plasma levels. The activation of the GH/IGF-1 axis leads to an increase of muscle mass and functional capacity. Ghrelin further acts on inflammation, appetite, and adipogenesis and for this reason was considered an important target to address catabolic conditions. We report the synthesis and properties of an indane based series of ghrelin receptor full agonists; they have been shown to generate a sustained increase of IGF-1 levels in dog and have been thoroughly investigated with respect to their functional activity.

Development of 2-pyrrolidinyl-N-methyl pyrimidones as potent and orally bioavailable HIV integrase inhibitors.

A series of 2-pyrrolidinyl-N-methyl pyrimidones HIV integrase inhibitors has been explored leading to the identification of derivative 13, which showed high activity at inhibiting viral replication in cell culture, favorable pharmacokinetic profile in two preclinical species, and an attractive profile against a panel of HIV-integrase mutants.

Enhancement of intestinal absorption of 2-methyl cytidine prodrugs.

The purpose of this study was to investigate the in vivo absorption enhancement of a nucleoside (phosphoramidate prodrug of 2'-methyl-cytidine) anti-viral agent of proven efficacy by means of intestinal permeation enhancers. Natural nucleosides are hydrophilic molecules that do not rapidly penetrate cell membranes by diffusion and their absorption relies on specialized transporters. Therefore, the oral absorption of nucleoside prodrugs and the target organ concentration of the biologically active nucleotide can be limited due to poor permeation across the intestinal epithelium. In the present study, the specificity, concentration dependence, and effect of four classes of absorption promoters, i.e. fatty acids, steroidal detergents, mucoadhesive polymers, and secretory transport inhibitors, were evaluated in a rat in vivo model. Sodium caprate and alpha-tocopheryl-polyethyleneglycol-1000-succinate (TPGS) showed a significant effect in increasing liver concentration of nucleotide (5-fold). These results suggested that both excipients might be suited in a controlled release matrix for the synchronous release of the drug and absorption promoter directly to the site of absorption and highlights that the effect is strictly dependent on the absorption promoter dose. The feasibility of such a formulation approach in humans was evaluated with the aim of developing a solid dosage form for the peroral delivery of nucleosides and showed that these excipients do provide a potential valuable tool in pre-clinical efficacy studies to drive discovery programs forward.

Phosphoramidate prodrugs of 2'-C-methylcytidine for therapy of hepatitis C virus infection.

The application of a phosphoramidate prodrug approach to 2'-C-methylcytidine (NM107), the first nucleoside inhibitor of the hepatitis C virus (HCV) NS5B polymerase, is reported. 2'-C-Methylcytidine, as its valyl ester prodrug (NM283), was efficacious in reducing the viral load in patients infected with HCV. Several of the phosphoramidates prepared demonstrated a 10- to 200-fold superior potency with respect to the parent nucleoside in the cell-based replicon assay. This is due to higher levels of 2'-C-methylcytidine triphosphate in the cells. These prodrugs are efficiently activated and converted to the triphosphate in hepatocytes of several species. Our SAR studies ultimately led to compounds that gave high levels of NTP in hamster and rat liver after subcutaneous dosing and that were devoid of the toxic phenol moiety usually found in ProTides.

Dihydroxy-pyrimidine and N-methylpyrimidone HIV-integrase inhibitors: improving cell based activity by the quaternarization of a chiral center.

In the context of HIV-integrase, dihydroxypyrimidine and N-methyl pyrimidone inhibitors the cellular activity of this class of compounds has been optimized by the introduction of a simple methyl substituent in the alpha-position of the C-2 side chains. Enhanced passive membrane permeability has been identified as the key factor driving the observed cell-based activity improvement. The rat PK profile of the alpha-methyl derivative 26a was also improved over its des-methyl exact analog.

Inhibitors of the hepatitis C virus NS3 protease with basic amine functionality at the P3-amino acid N-terminus: discovery and optimization of a new series of P2-P4 macrocycles.

In a follow-up to our recent disclosure of P2-P4 macrocyclic inhibitors of the hepatitis C virus (HCV) NS3 protease (e.g., 1, Chart 1), we report a new but related compound series featuring a basic amine at the N-terminus of the P3-amino acid residue. Replacement of the electroneutral P3-amino acid capping group (which is a feature of almost all tripeptide-like inhibitors of NS3 reported to date) with a basic group is not only tolerated but can result in advantageous cell based potency. Optimization of this new class of P3-amine based inhibitors gave compounds such as 25 and 26 that combine excellent cell based activity with pharmacokinetic properties that are attractive for an antiviral targeting HCV.

Cyclic phosphoramidates as prodrugs of 2'-C-methylcytidine.

The currently approved treatment for hepatitis C virus infections is a combination of Ribavirin and pegylated Interferon. It leads to a sustained virologic response in approximately only half of the patients treated. For this reason there is an urgent need of new therapeutic agents. 2'-C-Methylcytidine is the first nucleoside inhibitor of the HCV NS5B polymerase that was efficacious in reducing the viral load in patients infected with HCV. The application of a monophosphate prodrug approach based on unprecedented cyclic phosphoramidates is reported. Our SAR studies led to compounds that are efficiently converted to the active triphosphate in human hepatocytes.

Synthesis and evaluation of novel phosphoramidate prodrugs of 2'-methyl cytidine as inhibitors of hepatitis C virus NS5B polymerase.

A variety of new prodrugs of 2'-methyl cytidine based on acyloxy ethylamino phosphoramidates have been synthesized and tested in vitro and in vivo for their biological activity. Compared with the parent drug a 10- to 20-fold increase in formation of nucleotide triphosphate in rat and human hepatocytes could be achieved.

Discovery of raltegravir, a potent, selective orally bioavailable HIV-integrase inhibitor for the treatment of HIV-AIDS infection.

Human immunodeficiency virus type-1 (HIV-1) integrase is one of the three virally encoded enzymes required for replication and therefore a rational target for chemotherapeutic intervention in the treatment of HIV-1 infection. We report here the discovery of Raltegravir, the first HIV-integrase inhibitor approved by FDA for the treatment of HIV infection. It derives from the evolution of 5,6-dihydroxypyrimidine-4-carboxamides and N-methyl-4-hydroxypyrimidinone-carboxamides, which exhibited potent inhibition of the HIV-integrase catalyzed strand transfer process. Structural modifications on these molecules were made in order to maximize potency as HIV-integrase inhibitors against the wild type virus, a selection of mutants, and optimize the selectivity, pharmacokinetic, and metabolic profiles in preclinical species. The good profile of Raltegravir has enabled its progression toward the end of phase III clinical trials for the treatment of HIV-1 infection and culminated with the FDA approval as the first HIV-integrase inhibitor for the treatment of HIV-1 infection.

Design and synthesis of bicyclic pyrimidinones as potent and orally bioavailable HIV-1 integrase inhibitors.

HIV integrase is one of the three enzymes encoded by HIV genome and is essential for viral replication, but integrase inhibitors as marketed drugs have just very recently started to emerge. In this study, we show the evolution from the N-methylpyrimidinone structure to bicyclic pyrimidinones. Introduction of a suitably substituted amino moiety modulated the physical-chemical properties of the molecules and conferred nanomolar activity in the inhibition of spread of HIV-1 infection in cell culture. An extensive SAR study led to sulfamide (R)- 22b, which inhibited the strand transfer with an IC50 of 7 nM and HIV infection in MT4 cells with a CIC95 of 44 nM, and ketoamide (S)- 28c that inhibited strand transfer with an IC50 of 12 nM and the HIV infection in MT4 cells with a CIC95 of 13 nM and exhibited a good pharmacokinetic profile when dosed orally to preclinical species.

Discovery and synthesis of HIV integrase inhibitors: development of potent and orally bioavailable N-methyl pyrimidones.

The human immunodeficiency virus type-1 (HIV-1) encodes three enzymes essential for viral replication: a reverse transcriptase, a protease, and an integrase. The latter is responsible for the integration of the viral genome into the human genome and, therefore, represents an attractive target for chemotherapeutic intervention against AIDS. A drug based on this mechanism has not yet been approved. Benzyl-dihydroxypyrimidine-carboxamides were discovered in our laboratories as a novel and metabolically stable class of agents that exhibits potent inhibition of the HIV integrase strand transfer step. Further efforts led to very potent compounds based on the structurally related N-Me pyrimidone scaffold. One of the more interesting compounds in this series is the 2-N-Me-morpholino derivative 27a, which shows a CIC95 of 65 nM in the cell in the presence of serum. The compound has favorable pharmacokinetic properties in three preclinical species and shows no liabilities in several counterscreening assays.

Dihydroxypyrimidine-4-carboxamides as novel potent and selective HIV integrase inhibitors.

Human immunodeficiency virus type-1 (HIV-1) integrase, one of the three constitutive viral enzymes required for replication, is a rational target for chemotherapeutic intervention in the treatment of AIDS that has also recently been confirmed in the clinical setting. We report here on the design and synthesis of N-benzyl-5,6-dihydroxypyrimidine-4-carboxamides as a class of agents which exhibits potent inhibition of the HIV-integrase-catalyzed strand transfer process. In the current study, structural modifications on these molecules were made in order to examine effects on HIV-integrase inhibitory potencies. One of the most interesting compounds for this series is 2-[1-(dimethylamino)-1-methylethyl]-N-(4-fluorobenzyl)-5,6-dihydroxypyrimidine-4-carboxamide 38, with a CIC95 of 78 nM in the cell-based assay in the presence of serum proteins. The compound has favorable pharmacokinetic properties in preclinical species (rats, dogs, and monkeys) and shows no liabilities in several counterscreening assays, highlighting its potential as a clinically useful antiviral agent.

4,5-dihydroxypyrimidine carboxamides and N-alkyl-5-hydroxypyrimidinone carboxamides are potent, selective HIV integrase inhibitors with good pharmacokinetic profiles in preclinical species.

The dihydroxypyrimidine carboxamide 4a was discovered as a potent and selective HIV integrase strand transfer inhibitor. The optimization of physicochemical properties, pharmacokinetic profiles, and potency led to the identification of 13 in the dihydroxypyrimidine series and 18 in the N-methylpyrimidinone series having low nanomolar activity in the cellular HIV spread assay in the presence of 50% normal human serum and very good pharmacokinetics in preclinical species.

The design and enzyme-bound crystal structure of indoline based peptidomimetic inhibitors of hepatitis C virus NS3 protease.

The design of a series of peptidomimetic inhibitors of the hepatitis C virus NS3 protease is described. These inhibitors feature an indoline-2-carboxamide as a novel heterocyclic replacement for the P3 amino acid residue and N-terminal capping group of tripeptide based inhibitors. The crystal structure of the ternary NS3/NS4A/inhibitor complex for the most active molecule in this series highlights its suitability as an N-terminal capping group of a dipeptide inhibitor of the NS3 protease.

Capped dipeptide phenethylamide inhibitors of the HCV NS3 protease.

The N-terminal aminoacid of phenethylamide tripeptide inhibitors of the hepatitis C virus NS3 protease can be replaced with an alpha-hydroxy acid to obtain more 'drug like' inhibitors with low micromolar activity. The preferred S-configuration of the capping residue can be explained by molecular modeling studies.

Enantioselective total synthesis of the antitumor macrolide rhizoxin D.

The convergent, highly enantioselective synthesis of rhizoxin D, a natural product possessing potent antitumor and antifungal bioactivity, is described. The C(1)-C(9) fragment of the molecule was synthesized utilizing a threefold pseudosymmetric intermediate ultimately derived from gamma-butyrolactone. The central core of rhizoxin D was prepared via a chiral resolution/asymmetric aldol protocol. Several methods for the generation of the polyene fragment were explored, and the side-chain was ultimately prepared from serine in six steps. The unification of the left and right wings of the molecule was achieved using a one-step olefination protocol, and the macrocyclization was carried out using a Horner-Emmons olefination at the C(2)-C(3) olefin.

Capped dipeptide alpha-ketoacid inhibitors of the HCV NS3 protease.

The N-terminal aminoacid of alpha-ketotripeptide inhibitors of the hepatitis C virus NS3 protease can be replaced with an alpha-hydroxy acid, leading to capped dipeptide inhibitors such as 20 with an IC(50) value of 3.0 microM. The importance of the lipophilic side chain interactions at S3 of the protease and the requirement of the capping residue with R configuration have been explained by molecular modeling studies.