Complete measurement of the pKa values of the carboxyl and imidazole groups in Bacillus circulans xylanase.

Electrostatic interactions in proteins can be dissected experimentally by determining the pKa values of their constituent ionizable amino acids. To complement previous studies of the glutamic acid and histidine residues in Bacillus circulans xylanase (BCX), we have used NMR methods to measure the pKa s of the seven aspartic acids and the C-terminus of this protein. The pKa s of these carboxyls are all less than the corresponding values observed with random coil polypeptides, indicating that their ionization contributes favorably to the stability of the folded enzyme. In general, the aspartic acids with the most reduced pKa s are those with limited exposure to the solvent and a high degree of conservation among homologous xylanases. Most dramatically, Asp 83 and Asp 101 have pKa s < 2 and thus remain deprotonated in native BCX under all conditions examined. Asp 83 is completely buried, forming a strong salt bridge with Arg 136. In contrast, Asp 101 is located on the surface of the protein, stabilized in the deprotonated form by an extensive network of hydrogen bonds involving an internal water molecule and the neutral side-chain and main-chain atoms of Ser 100 and Thr 145. These data provide a complete experimental database for theoretical studies of the ionization behavior of BCX under acidic conditions.     

Tricyclic imidazole antagonists of the Neuropeptide S Receptor

A new structural class of potent antagonists of the Neuropeptide S Receptor (NPSR) is reported. High-throughput screening identified a tricyclic imidazole antagonist of NPSR, and medicinal chemistry optimization of this structure was undertaken to improve potency against the receptor as well as CNS penetration. Detailed herein are synthetic and medicinal chemistry studies that led to the identification of antagonists 15 and NPSR-PI1, which demonstrate potent in vitro NPSR antagonism and central exposure in vivo.     

Orally bioavailable,liver-selective stearoyl-CoA desaturase (SCD) inhibitors

We discovered a structurally novel SCD (Δ9 desaturase) inhibitor 4a (CVT-11,563) that has 119 nM potency in a human cell-based (HEPG2) SCD assay and selectivity against Δ5 and Δ6 desaturases. This compound has 90% oral bioavailability (rat) and excellent plasma exposure (dAUC 935 ng h/mL). Additionally, 4a shows moderately selective liver distribution (three times vs plasma and adipose tissue) and relatively low brain penetration. In a five-day study (high sucrose diet, rat) compound 4a significantly reduced SCD activity as determined by GC analysis of fatty acid composition in plasma and liver. We describe the discovery of 4a from HTS hit 1 followed by scaffold replacement and SAR studies focused on DMPK properties.     

The design,synthesis and structure-activity relationships associated with C28 amine-based betulinic acid derivatives as inhibitors of HIV-1 maturation

The design and synthesis of a series of C28 amine-based betulinic acid derivatives as HIV-1 maturation inhibitors is described. This series represents a continuation of efforts following on from previous studies of C-3 benzoic acid-substituted betulinic acid derivatives as HIV-1 maturation inhibitors (MIs) that were explored in the context of C-28 amide substituents. Compared to the C-28 amide series, the C-28 amine derivatives exhibited further improvements in HIV-1 inhibitory activity toward polymorphisms in the Gag polyprotein as well as improved activity in the presence of human serum. However, plasma exposure of basic amines following oral administration to rats was generally low, leading to a focus on moderating the basicity of the amine moiety distal from the triterpene core. The thiomorpholine dioxide (TMD) 20 emerged from this study as a compound with the optimal antiviral activity and an acceptable pharmacokinetic profile in the C-28 amine series. Compared to the C-28 amide 3, 20 offers a 2- to 4-fold improvement in potency towards the screening viruses, exhibits low shifts in the EC₅₀ values toward the V370A and ΔV370 viruses in the presence of human serum or human serum albumin, and demonstrates improved potency towards the polymorphic T371A and V362I virus variants.     

Synthesis and pharmacological characterization of functionalized 6-piperazin-1-yl-purines as cannabinoid receptor 1 (CB1) inverse agonists

Antagonists of peripheral type 1 cannabinoid receptors (CB1) may have utility in the treatment of obesity, liver disease, metabolic syndrome and dyslipidemias. We have targeted analogues of the purine inverse agonist otenabant (1) for this purpose. The non-tissue selective CB1 antagonist rimonabant (2) was approved as a weight-loss agent in Europe but produced centrally mediated adverse effects in some patients including dysphoria and suicidal ideation leading to its withdrawal. Efforts are now underway to produce compounds with limited brain exposure. While many structure-activity relationship (SAR) studies of 2 have been reported, along with peripheralized compounds, 1 remains relatively less studied. In this report, we pursued analogues of 1 in which the 4-aminopiperidine group was switched to piperazine group to enable a better understanding of SAR to eventually produce compounds with limited brain penetration. To access a binding pocket and modulate physical properties, the piperazine was functionalized with alkyl, heteroalkyl, aryl and heteroaryl groups using a variety of connectors, including amides, sulfonamides, carbamates and ureas. These studies resulted in compounds that are potent antagonists of hCB1 with high selectivity for hCB1 over hCB2. The SAR obtained led to the discovery of 65 (Ki?=?4?nM, >1,000-fold selective for hCB1 over hCB2), an orally bioavailable aryl urea with reduced brain penetration, and provides direction for discovering peripherally restricted compounds with good in vitro and in vivo properties.     

Discovery and characterization of [(cyclopentyl)ethyl]benzoic acid inhibitors of microsomal prostaglandin E synthase-1

We describe a novel class of acidic mPGES-1 inhibitors with nanomolar enzymatic and human whole blood (HWB) potency. Rational design in conjunction with structure-based design led initially to the identification of anthranilic acid 5, an mPGES-1 inhibitor with micromolar HWB potency. Structural modifications of 5 improved HWB potency by over 1000×, reduced CYP2C9 single point inhibition, and improved rat clearance, which led to the selection of [(cyclopentyl)ethyl]benzoic acid compound 16 for clinical studies. Compound 16 showed an IC₈₀ of 24 nM for inhibition of PGE₂ formation in vitro in LPS-stimulated HWB. A single oral dose resulted in plasma concentrations of 16 that exceeded its HWB IC₈₀ in both rat (5 mg/kg) and dog (3 mg/kg) for over twelve hours.     

From a novel HTS hit to potent,selective, and orally bioavailable KDM5 inhibitors

A high-throughput screening (HTS) of the Genentech/Roche library identified a novel, uncharged scaffold as a KDM5A inhibitor. Lacking insight into the binding mode, initial attempts to improve inhibitor potency failed to improve potency, and synthesis of analogs was further hampered by the presence of a C–C bond between the pyrrolidine and pyridine. Replacing this with a C–N bond significantly simplified synthesis, yielding pyrazole analog 35, of which we obtained a co-crystal structure with KDM5A. Using structure-based design approach, we identified 50 with improved biochemical, cell potency and reduced MW and lower lipophilicity (Log D) compared with the original hit. Furthermore, 50 showed lower clearance than 9 in mice. In combination with its remarkably low plasma protein binding (PPB) in mice (40%), oral dosing of 50 at 5 mg/kg resulted in unbound C_max ～2-fold of its cell potency (PC9 H3K4Me3 0.96 μM), meeting our criteria for an in vivo tool compound from a new scaffold.     

Peripherally restricted CB1 receptor blockers

Antagonists (inverse agonists) of the cannabinoid-1 (CB1) receptor showed promise as new therapies for controlling obesity and related metabolic function/liver disease. These agents, representing diverse chemical series, shared the property of brain penetration due to the initial belief that therapeutic benefit was mainly based on brain receptor interaction. However, undesirable CNS-based side effects of the only marketed agent in this class, rimonabant, led to its removal, and termination of the development of other clinical candidates soon followed. Re-evaluation of this approach has focused on neutral or peripherally restricted (PR) antagonists. Supporting these strategies, pharmacological evidence indicates most if not all of the properties of globally acting agents may be captured by molecules with little brain presence. Methodology that can be used to eliminate BBB penetration and the means (in vitro assays, tissue distribution and receptor occupancy determinations, behavioral paradigms) to identify potential agents with little brain presence is discussed. Focus will be on the pharmacology supporting the contention that reported agents are truly peripherally restricted. Notable examples of these types of compounds are: TM38837 (structure not disclosed); AM6545 (8); JD5037 (15b); RTI-12 (19).     

Optimization of allosteric MEK inhibitors. Part 1: Venturing into underexplored SAR territories

Using PD325901 as a starting point for identifying novel allosteric MEK inhibitors with high cell potency and long-lasting target inhibition in vivo, truncation of its hydroxamic ester headgroup was combined with incorporation of alkyl and aryl ethers at the neighboring ring position. Whereas alkoxy side chains did not yield sufficient levels of cell potency, specifically substituted aryloxy groups allowed for high enzymatic and cellular potencies. Sulfamide 28 was identified as a highly potent MEK inhibitor with nanomolar cell potency against B-RAF (V600E) as well as Ras-mutated cell lines, high metabolic stability and resulting long half-lives. It was efficacious against B-RAF as well as K-Ras driven xenograft models and showed—despite being orally bioavailable and not a P-glycoprotein substrate—much lower brain/plasma exposure ratios than PD325901.     

Calculating pKa values in enzyme active sites

The ionization properties of the active-site residues in enzymes are of considerable interest in the study of the catalytic mechanisms of enzymes. Knowledge of these ionization constants (pKa values) often allows the researcher to identify the proton donor and the catalytic nucleophile in the reaction mechanism of the enzyme. Estimates of protein residue pKa values can be obtained by applying pKa calculation algorithms to protein X-ray structures. We show that pKa values accurate enough for identifying the proton donor in an enzyme active site can be calculated by considering in detail only the active-site residues and their immediate electrostatic interaction partners, thus allowing for a large decrease in calculation time. More specifically we omit the calculation of site-site interaction energies, and the calculation of desolvation and background interaction energies for a large number of pairs of titratable groups. The method presented here is well suited to be applied on a genomic scale, and can be implemented in most pKa calculation algorithms to give significant reductions in calculation time with little or no impact on the accuracy of the results. The work presented here has implications for the understanding of enzymes in general and for the design of novel biocatalysts.     

Identification of substituted benzothiazole sulfones as potent and selective inhibitors of endothelial lipase

A low level of high density lipoprotein (HDL) is an independent risk factor for cardiovascular disease. HDL reduces inflammation and plays a central role in reverse cholesterol transport, where cholesterol is removed from peripheral tissues and atherosclerotic plaque. One approach to increase plasma HDL is through inhibition of endothelial lipase (EL). EL hydrolyzes phospholipids in HDL resulting in reduction of plasma HDL. A series of benzothiazole sulfone amides was optimized for EL inhibition potency, lipase selectivity and improved pharmacokinetic profile leading to the identification of Compound 32. Compound 32 was evaluated in a mouse pharmacodynamic model and found to show no effect on HDL cholesterol level despite achieving targeted plasma exposure (C_trough > 15 fold over mouse plasma EL IC₅₀ over 4 days).     

Discovery of novel chiral diazepines as bombesin receptor subtype-3 (BRS-3) agonists with low brain penetration

The discovery and optimization of a novel series of BRS-3 agonists are described. We explored a potent BRS-3 agonist with low brain penetration to avoid an adverse effect derived from central nervous system exposure. Through the derivatization process, chiral diazepines 9f and 9g were identified as possessing low brain penetration as well as potent in vitro activity against human and mouse BRS-3s.     

Discovery and structure–activity relationship of 1,3-cyclohexyl amide derivatives as novel mGluR5 negative allosteric modulators

A novel series of trans-1,3-cyclohexyl diamides was discovered and characterized as mGluR5 negative allosteric modulators (NAMs) lacking an alkyne moiety. Conformational constraint of one of the amide bonds in the diamide template led to a spirooxazoline template. A representative compound (24d) showed good in vitro potency, high CNS penetration and, upon subcutaneous dosing, demonstrated efficacy in the mouse marble burying test, generally used as indicative of potential anxiolytic activity.     

Design and synthesis of HIV-1 protease inhibitors for a long-acting injectable drug application

The design and synthesis of novel HIV-1 protease inhibitors (PIs) (1–22), which display high potency against HIV-1 wild-type and multi-PI-resistant HIV-mutant clinical isolates, is described. Lead optimization was initiated from compound 1, a Phe–Phe hydroxyethylene peptidomimetic PI, and was directed towards the discovery of new PIs suitable for a long-acting (LA) injectable drug application. Introducing a heterocyclic 6-methoxy-3-pyridinyl or a 6-(dimethylamino)-3-pyridinyl moiety (R³) at the para-position of the P1′ benzyl fragment generated compounds with antiviral potency in the low single digit nanomolar range. Halogenation or alkylation of the metabolic hot spots on the various aromatic rings resulted in PIs with high stability against degradation in human liver microsomes and low plasma clearance in rats. Replacing the chromanolamine moiety (R¹) in the P2 protease binding site by a cyclopentanolamine or a cyclohexanolamine derivative provided a series of high clearance PIs (16–22) with EC₅₀s on wild-type HIV-1 in the range of 0.8–1.8 nM. PIs 18 and 22, formulated as nanosuspensions, showed gradual but sustained and complete release from the injection site over two months in rats, and were therefore identified as interesting candidates for a LA injectable drug application for treating HIV/AIDS.     

Discovery of aryl ureas and aryl amides as potent and selective histamine H3 receptor antagonists for the treatment of obesity (Part II)

A novel series of histamine H₃ receptor (H₃R) antagonists was derived from an arylurea lead series (1) via bioisosteric replacement of the urea functionality by an amide linkage. The arylamide series was optimized through SAR studies by a broad variation of substituents in the left-hand side benzoyl residue (analogs 2a–2ag) or replacement of the benzoyl moiety by heteroarylcarbonyl residues (analogs 5a–5n). Compounds 2p and 2q were identified within the series as potent and selective H₃R antagonists/inverse agonists with acceptable overall profile. Compound 2q was orally active in food intake inhibition in diet-induced obese (DIO) mice. Compound 2q represents a novel H₃R antagonist template with improved in vitro potency and oral efficacy and has its merits as a new lead for further optimization.     

Pyrazole antagonists of the CB1 receptor with reduced brain penetration

Type 1 cannabinoid receptor (CB1) antagonists might be useful for treating obesity, liver disease, metabolic syndrome, and dyslipidemias. Unfortunately, inhibition of CB1 in the central nervous system (CNS) produces adverse effects, including depression, anxiety and suicidal ideation in some patients, which led to withdrawal of the pyrazole inverse agonist rimonabant (SR141716A) from European markets. Efforts are underway to produce peripherally selective CB1 antagonists to circumvent CNS-associated adverse effects. In this study, novel analogs of rimonabant (1) were explored in which the 1-aminopiperidine group was switched to a 4-aminopiperidine, attached at the 4-amino position (5). The piperidine nitrogen was functionalized with carbamates, amides, and sulfonamides, providing compounds that are potent inverse agonists of hCB1 with good selectivity for hCB1 over hCB2. Select compounds were further studied using in vitro models of brain penetration, oral absorption and metabolic stability. Several compounds were identified with predicted minimal brain penetration and good metabolic stability. In vivo pharmacokinetic testing revealed that inverse agonist 8c is orally bioavailable and has vastly reduced brain penetration compared to rimonabant.     

Identification of N-(2-(azepan-1-yl)-2-phenylethyl)-benzenesulfonamides as novel inhibitors of GlyT1

A novel series of glycine transporter 1 (GlyT1) inhibitors is described. Scoping of the heterocycle moiety of hit 4-chlorobenzenesulfonamide 1 led to replacement of the piperidine with an azepane for a modest increase in potency. Phenyl sulfonamides proved superior to alkyl and non-phenyl aromatic sulfonamides, while subsequent ortho substitution of the 2-(azepan-1-yl)-2-phenylethanamine aromatic ring yielded 39 (IC₅₀ 37 nM, solubility 14 μM), the most potent GlyT1 inhibitor in this series. Favorable brain–plasma ratios were observed for select compounds in pharmacokinetic studies to evaluate CNS penetration.     

Identification and optimization of indolo[2,3-c]quinoline inhibitors of IRAK4

IRAK4 is responsible for initiating signaling from Toll-like receptors (TLRs) and members of the IL-1/18 receptor family. Kinase-inactive knock-ins and targeted deletions of IRAK4 in mice cause reductions in TLR induced pro-inflammatory cytokines and these mice are resistant to various models of arthritis. Herein we report the identification and optimization of a series of potent IRAK4 inhibitors. Representative examples from this series showed excellent selectivity over a panel of kinases, including the kinases known to play a role in TLR-mediated signaling. The compounds exhibited low nM potency in LPS- and R848-induced cytokine assays indicating that they are blocking the TLR signaling pathway. A key compound (26) from this series was profiled in more detail and found to have an excellent pharmaceutical profile as measured by predictive assays such as microsomal stability, TPSA, solubility, and c log P. However, this compound was found to afford poor exposure in mouse upon IP or IV administration. We found that removal of the ionizable solubilizing group (32) led to increased exposure, presumably due to increased permeability. Compounds 26 and 32, when dosed to plasma levels corresponding to ex vivo whole blood potency, were shown to inhibit LPS-induced TNFα in an in vivo murine model. To our knowledge, this is the first published in vivo demonstration that inhibition of the IRAK4 pathway by a small molecule can recapitulate the phenotype of IRAK4 knockout mice.     

Structural and computational basis for potent inhibition of glutamate carboxypeptidase II by carbamate-based inhibitors

A series of carbamate-based inhibitors of glutamate carboxypeptidase II (GCPII) were designed and synthesized using ZJ-43, N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-l-glutamic acid, as a molecular template in order to better understand the impact of replacing one of the two nitrogen atoms in the urea-based GCPII inhibitor with an oxygen atom. Compound 7 containing a C-terminal 2-oxypentanedioic acid was more potent than compound 5 containing a C-terminal glutamic acid (2-aminopentanedioic acid) despite GCPII’s preference for peptides containing an N-terminal glutamate as substrates. Subsequent crystallographic analysis revealed that ZJ-43 and its two carbamate analogs 5 and 7 with the same (S,S)-stereochemical configuration adopt a nearly identical binding mode while (R,S)-carbamate analog 8 containing a d-leucine forms a less extensive hydrogen bonding network. QM and QM/MM calculations have identified no specific interactions in the GCPII active site that would distinguish ZJ-43 from compounds 5 and 7 and attributed the higher potency of ZJ-43 and compound 7 to the free energy changes associated with the transfer of the ligand from bulk solvent to the protein active site as a result of the lower ligand strain energy and solvation/desolvation energy. Our findings underscore a broader range of factors that need to be taken into account in predicting ligand-protein binding affinity. These insights should be of particular importance in future efforts to design and develop GCPII inhibitors for optimal inhibitory potency.     

Second basic pKa: An overlooked parameter in predicting phospholipidosis-inducing potential of diamines

In this paper, we present the phospholipidosis-inducing potential (PLIP) of forty fragment-sized diamines derived from N-benzyl-4-(methylamino)piperidine and discuss the relationship between their PLIP and the physicochemical properties. Our results demonstrate that the previously reported methods are not suitable for predicting the PLIP of fragment-sized diamines; the second basic pKa can distinguish PLIP-positive diamines from PLIP-negative diamines more accurately than ClogP or most basic pKa. To the best of our knowledge, this is the first report describing the relationship between PLIP and second basic pKa.