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Through medicinal chemistry lead optimization studies focused on calculated properties and guided by X-ray crystallography and computational modeling, potent pan-JNK inhibitors were identified that showed submicromolar activity in a cellular assay. Using in vitro ADME profiling data, 9t was identified as possessing favorable permeability and a low potential for efflux, but it was rapidly cleared in liver microsomal incubations. In a mouse pharmacokinetics study, compound 9t was brain-penetrant after oral dosing, but exposure was limited by high plasma clearance. Brain exposure at a level expected to support modulation of a pharmacodynamic marker in mouse was achieved when the compound was coadministered with the pan-cytochrome P450 inhibitor 1-aminobenzotriazole.

A novel series of cyclic urea-based CCR5 antagonists was designed aiming to resolve instability issue in the fasted simulated intestinal fluid (FSIF) associated with the acyclic urea moiety in 1. This class of CCR5 compounds demonstrated high antiviral activities against HIV-1 infection in both HOS and PBL assays. Further evaluation of these compounds indicated that 16-R not only substantially enhanced its stability, but also exhibited excellent pharmacokinetics properties.

We describe the synthesis and potency of a novel series of N-substituted 2-phenyl- and 2-methyl-2-phenyl-1,4-diaminobutane- based CCR5 antagonists. Compounds 7a and 12f were found to be potent in anti-HIV assays and bioavailable in the low-dose rat PK model.

Modification of the acyl moiety in the CCR5 lead molecule 2 led to identification of several new classes of CCR5 antagonists. Antiviral activity and pharmacokinetic properties of the synthesized compounds were evaluated. Structure-activity relationship (SAR) derived from these studies further guided the optimization efforts, ultimately leading to the discovery of 36 with an acceptable drug-like profile.

Using AMD070 as a starting point for structural modification, a novel series of isoquinoline CXCR4 antagonists was developed. A structure-activity scan of alternate lower heterocycles led to the 3-isoquinolinyl moiety as an attractive replacement for benzimidazole. Side chain optimization in the isoquinoline series led to a number of compounds with low nanomolar anti-HIV activities and promising rat PK properties.

The lead optimization of a series of N-substituted benzimidazole CXCR4 antagonists is described. Side chain modifications and stereochemical optimization led to substantial improvements in potency and protein shift to afford compounds with low nanomolar anti-HIV activity.

Synthesis of several novel imidazopyridine-5,6,7,8-tetrahydro-8-quinolinamine derivatives with potent activity against HIV are described. Synthetic approaches allowing for variation of the substitution pattern are outlined and resulting changes in antiviral activity and pharmacokinetics are highlighted. Several compounds with low nanomolar anti-HIV activity and oral bioavailability are described.

Several series of CCR5 antagonists have been discovered by derivatization at the N-terminal of the piperidine ring of the core template 2. Some derivatives exhibited potent inhibition against HIV-1infection. The pharmacokinetic properties of the lead compounds 11a, 14a, 15b, and 16b have been evaluated in vivo.

We describe robust chemical approaches toward putative CCR5 scaffolds designed in our laboratories. Evaluation of analogues in the (125)I-[MIP-1beta] binding and Ba-L-HOS antiviral assays resulted in the discovery of 64 and 68 in the 4,4-disubstitited piperidine class H, both potent CCR5 ligands (pIC 50 = 8.30 and 9.00, respectively) and HIV-1 inhibitors (pIC 50 = 7.80 and 7.84, respectively, in Ba-L-HOS assay). In addition, 64 and 68 were bioavailable in rodents, establishing them as lead molecules for further optimization toward CCR5 clinical candidates.

The metabolism of prostaglandin E2 (PGE2) in isolated rat hepatocytes led to the formation of four major as well as several minor products which were structurally characterized using electrospray tandem mass spectrometry. The major metabolites identified included dinor-PGE1, dinor-PGE2, and tetranor-PGE1 and the taurine conjugates of dinor-PGE1 and dinor-PGE2. Several minor metabolites including the taurine conjugates of PGE2 and tetranor PGE1 along with a glucuronide conjugate of PGE2 were also identified. These taurine conjugates had not been previously identified in studies of PGE2 metabolism, yet comprised nearly 50% of the mixture of metabolites after 40-min incubations. Experiments carried out with deuterium-labeled PGE2 ([3,3,4,4-D4]PGE2) resulted in the complete loss of all deuterium atoms in dinor-PGE1, dinor-PGE2, and tetranor metabolites during incubation with hepatocytes. Metabolism via classic beta-oxidation pathways would predict one deuterium atom retained by dinor-PGE1 and two deuterium atoms retained by dinor-PGE2. When PGE2 was incubated with isolated rat hepatocytes in buffer containing 30% D2O, substantial incorporation (30%) of one deuterium atom could be observed in the dinor metabolites along with 10% incorporation into the tetranor and residual PGE2. Deuterium-labeled PGE1 ([3,3,4,4-D4]PGE1) was metabolized to D2-dinor-PGE1, tetranor-PGE1, and the taurine conjugate of D2-dinor-PGE1 by isolated rat hepatocytes. The loss of deuterium during metabolism of the deuterated substrates of PGE2, but not PGE1, as well as the incorporation of deuterium atoms from the aqueous solvent into PGE2 metabolites suggested that the delta 5 double bond and sequential isomerization reactions lead to eventual exchange of the protons from carbon atom 4 of PGE2 with water.

New stable leukotriene A 4 analog with acetylenic function in positions 14,15 has been synthesized. Conversion of this analog to the corresponding LTC 4 derivative and the structural and biological characterization are presented.

A reverse-phase–HPLC/tandem mass spectrometric method (LC/MS/MS) was developed for the quantitation of leukotriene B 4 (LTB 4 ), the 5-lipoxygenase product, 5-hydroxyeicosatetraenoic acid (5-HETE), as well as the ω-oxidation metabolites of LTB 4 , 20-hydroxy-LTB 4 , and 20-carboxy-LTB 4 . Electrospray-generated carboxylate anions were collisionally activated and decomposed to specific and abundant product ions and multiple reaction monitoring was used to analyze LTB 4 ( m / z 335 → 195), 20-hydroxy-LTB 4 ( m / z 351 → 195), 20-carboxy-LTB 4 ( m / z 365 → 195), and 5-HETE ( m / z 319 → 115). A linear correlation was observed in comparison of results obtained from quantitation by LC/MS/MS with quantitation by gas chromatography/MS of the pentafluorobenzyl ester/trimethylsilyl ether derivative of LTB 4 produced during opsonized zymosan stimulation of human neutrophils. Detection limits at the low picogram level were obtained for all metabolites. This method was applied to the quantitation of 5-lipoxygenase products produced from zymosan-stimulated human neutrophils in the presence of ethanol. At physiologically relevant concentrations of ethanol, production of all 5-lipoxygenase products generated by stimulated neutrophils was markedly attenuated.

Analysis by gas chromatography/mass spectrometry (GC/MS) of derivatized metabolites formed following incubation of leukotriene B4 (LTB4) incubation with Ito cells extends previous knowledge concerning fragmentation mechanisms for derivatized hydroxy-substituted unsaturated fatty acids. LTB4 was metabolized by rat Ito cells, a hepatic perisinusoidal stellate cell, by the delta 10- and delta 14-reductase pathways, resulting in the formation of 10,11-dihydro-LTB4 and 10,11,14,15-tetrahydro-LTB4. Formation of the intermediate metabolites, 12-oxo-10,11-dihydro-LTB4 and 12-oxo-10,11,14,15-tetrahydro-LTB4, was also observed. GC/electron impact (EI) MS analysis of the 12-oxo metabolites, derivatized as the pentafluorobenzyl ester/ trimethylsilyl ether compounds, resulted in unique fragmentations indicative of the oxo substituent and double bond positions. Further metabolism of 10,11-dihydro-LTB4 and 10,11,14,15-tetrahydro-LTB4 by carboxy terminus beta-oxidation resulted in chain-shortened monohydroxy metabolites. Possible intermediates in this metabolism, which resulted in loss of the original C-5 hydroxy substituent from LTB4, were identified as 2,4,6-conjugated triene-containing C-18 metabolites. The absence of a double bond allylic to the trimethylsiloxy ether in derivatized 10,11,14,15-tetrahydro LTB4 metabolites strikingly reduced the abundance of alpha-cleavage ions observed in the EI mass spectra of these compounds, thus suggesting the importance of formation of an allylic stabilized radical in such alpha-cleavage reactions. Lacking a favorable alpha-cleavage reaction, GC/EIMS analysis of 10-hydroxy-2,4,6-octadecatrienoic acid resulted in the formation of m/z 91, which may arise via cyclization of the conjugated triene moiety. In addition, GC/MS analysis of derivatized metabolites containing the 2,4,6 conjugated triene moiety resulted in a unique fragment ion in the electron capture ionization mass spectra that also may arise via cyclization of the conjugated triene with formation of m/z 121.

The low energy collision induced dissociation (CID) of the carboxylate anions generated by electrospray ionization of leukotriene B4 (LTB4) and 16 of its metabolites was studied in a tandem quadrupole mass spectrometer. LTB4 is a biologically active lipid mediator whose activity is terminated by metabolism into a wide variety of structural variants. The collision-induced dissociation spectra of the carboxylate anions revealed structurally informative ions whose formation was determined by the position of hydroxyl substituents and double bonds present in the LTB4 metabolite. Major ions resulted from charge remote α-hydroxy fragmentation or charge directed α-hydroxy fragmentation. The conjugated triene moiety present in some metabolites was proposed to undergo cyclization to a 1,3-cyclohexadiene structure prior to charge remote or charge driven a-hydroxy fragmentation. The mechanisms responsible for all major ions observed in the CID spectra were studied using stable isotope labeled analogs of the LTB4 metabolites. In general, the collision-induced decomposition of carboxylate anions produced unique spectra for all LTB4 derived metabolites. The observed decomposition product ions from the carboxylate anion could be useful in developing assays for these molecules in biological fluids.

The intermediate metabolic events which degrade hydroxy polyunsaturated fatty acids is largely unknown. Such molecules are common products of lipid peroxidation and lipoxygenase catalyzed oxidation of arachidonic acid. Metabolism of two 5,12-dihydroxyeicosatetraenoic acids, 6-trans-LTB4 (leukotriene B4), and 6-trans-12-epi-LTB4 was studied in HepG2 cells (a human-derived hepatoma cell line). Extensive metabolism was observed with a major metabolite identified as 4-hydroxy-6-dodecenoic acid for both epimers. Incubation of 6-trans-LTB4 epimers at shorter times revealed the formation of intermediate metabolites, including 6-hydroxy-4,8-tetradecadienoic acid and 8-hydroxy-4,6,10-hexadecatrienoic acid suggesting beta-oxidation as the major pathway leading to the formation of the common terminal metabolite. Two additional metabolites were structurally elucidated as 5-oxo-6,7-dihydro-LTB4 and 6,7-dihydro-LTB4 which have not been previously described. Formation of 5-oxo-6,7-dihydro-LTB4 and 6,7-dihydro-LTB4 were also observed during metabolism of 6-trans-12-epi-LTB4 in human polymorphonuclear leukocytes. Of particular interest is the metabolism of these compounds by beta-oxidation from the carboxyl terminus, a process which is not observed with leukotriene B4 or leukotriene C4. Identification of these metabolites suggested the operation of the 5-hydroxyeicosanoid dehydrogenase pathway followed by a delta 6-reductase metabolic pathway which has not been previously described. This pathway of beta-oxidation may limit the activity of various 5,12-diHETEs including nonenzymatic hydrolysis products of LTA4 and also the recently described B4-isoleukotrienes.

Design and practical syntheses of a novel class of cyclic urea-based CCR5-antagonists demonstrating high antiviral activities against HIV-1 infection in both HOS and PBL assays are described.

Long-chain acyl Coenzyme A (CoA) is essentially composed of three major chemical groups, fatty acyl-, phosphopantetheino-, and 3′,5′,-adenosine diphospho-moieties. The negative ion fast-atom bombardment mass spectrometry spectra of long-chain acyl CoA thioesters were characterized by the formation of abundant [M = H]− and two distinct classes of fragment ions, one class which retained the acyl group and another class which is related to CoA that contains the phosphopantethene and adenine. The ions which retained the acyl group in the spectrum of palmitoyl CoA appeared at m/z 675, 657, 595, and 577 and were found to decompose by loss of alkylketene observed at m/z 357 and 339. Those ions which retained the adenine group were observed at m/z 426 and 408. In contrast to these ions observed following fast-atom bombardment ionization, tandem mass spectrometry of the [M - H]−, from palmitoyl CoA (m/z 1004), yielded the adenine-containing ions as major products and the acyl-containing ions were of low abundance or not detected. These results suggested that the formation of many characteristic ions observed in direct FAB analysis occurred during the desorption process. The unique relationship between ions which involved the transition from acyl-containing ions to only CoA-containing ions by the loss of alkylketene allowed the development of tandem mass spectrometry protocols for the analysis of acyl CoA mixtures. Precursor scans of either m/z 357 or 339 yielded the identification of each species in a complex mixture. Identification of specific species was obtained with a neutral loss scan of the mass for a specific alkylketene.

A novel series of pyridyl carboxamide-based CCR5 inhibitors was designed, synthesized, and demonstrated to be highly potent against HIV-1 infection in both HOS and PBL assays. Attempts to evaluate this series of compounds in a rat PK model revealed its instability in rat plasma. A hypothesis for this liability was proposed, and strategies to overcome this issue were pursued, leading to discovery of highly potent 40 and 41, which featured dramatically improved rat PK profiles.

We recently described ( J. Med. Chem. 2008 , 51 , 6538 - 6546 ) a novel class of CCR5 antagonists with strong anti-HIV potency. Herein, we detail SAR converting leads 1 and 2 to druglike molecules. The pivotal structural motif enabling this transition was the secondary sulfonamide substituent. Further fine-tuning of the substituent pattern in the sulfonamide paved the way to enhancing potency and bioavailability and minimizing hERG inhibition, resulting in discovery of clinical compound 122 (GSK163929).

Six previously unidentified leukotriene (LT) B4 metabolites formed during incubation of LTB4 with human keratinocytes in primary culture indicate the importance of the 12-hydroxyeicosanoid dehydrogenase pathway in LTB4 metabolism. The ultraviolet absorption spectra obtained for all keratinocyte metabolites revealed the presence of a conjugated diene structural moiety rather than the conjugated triene structure of LTB4. Metabolites were characterized using fast atom bombardment-mass spectrometry, gas chromatography-mass spectrometry of the pentafluorobenzyl ester, trimethylsilyl ether derivatives and specific degradation reactions. The previously identified 10,11-dihydro-LTB4 and 10,11-dihydro-12-epi-LTB4 were observed as well as 20-OH-10,11-dihydro-LTB4, consistent with the reductase pathway of LTB4 metabolism. This pathway involves initial formation of 12-oxo-LTB4 catalyzed by 12-hydroxyeicosanoid dehydrogenase followed by reduction by delta 10-reductase. The most lipophilic metabolite of LTB4 was identified as 10-hydroxy-4,6,12-octadecatrienoic acid which could result from beta-oxidation reactions of LTB4 following reduction of the 10,11-double bond. One of the most abundant metabolites was characterized as 3,7,14-trihydroxy-8,10,16- docosatrienoic acid which could result from fatty acid elongation following reduction of the 10,11-double bond. Additional abundant LTB4 metabolites were identified that result from glutathione conjugation of 12-oxo-LTB4. These were characterized using fast atom bombardment-mass spectrometry and by chemical degradation using hypochlorous acid as well as transpeptidases. These metabolites were identified as 5,12-dihydroxy-6-glutathionyl-7,9,14-eicosatrienoic acid (c-LTB3), 5,12-dihydroxy-6-cysteinyl-glycyl-7,9,14-eicosatrienoic acid (d-LTB3) and 5,12-dihydroxy-6-cysteinyl-7,9,14-eicosatrienoic acid (e-LTB3). We propose that these metabolites result from a 1,8 Michael-type addition of glutathione to the 12-oxo-LTB4 intermediate.

The low-energy collision-induced dissociation (CID) of the carboxylate anions generated by fast atom bombardment ionization of monohydroxy unsaturated fatty acids derived from oleic, linoleic, linolenic and arachidonic acids were studied in a tandem quadrupole mass spectrometer. The collisional activation spectra revealed structurally informative ions as to the position of the hydroxyl substituent in relationship to the sites of unsaturation. Five mechanisms are proposed for the fragmentation of hydroxyl substituted unsaturated fatty acids and are dependent upon the presence of alpha- or beta-unsaturation sites. These mechanisms include charge-remote allylic fragmentation, charge-remote vinylic fragmentation, charge-driven allylic fragmentation, charge-driven vinylic fragmentation, and homolytic fragmentation by an oxy-Cope rearrangement process. The assignment of specific fragmentation pathways was supported in many instances with deuterium-labeled analogs. Although no single fragmentation mechanism appears to predominate, a rational approach to the interpretation of these CID spectra is proposed. The CID spectra of unknown compounds could be used to establish the hydroxyl substituent position in relationship to certain sites of unsaturation but would not be indicative of all double bond locations. The oxy-Cope rearrangement is specific for a structural unit, namely the 3-hydroxy-1,5-diene moiety.

Several series of carbamate, urea and carboxamide-based CCR5 antagonists have been discovered via optimizations at the amine portion of lead compound 2. All compounds were evaluated for their antiviral activities. Lead urea 29 showed good pharmacokinetic properties, justifying further development of this series.

Production of platelet-activating factor (PAF) during opsonized zymosan stimulation of human polymorphonuclear leukocytes is dependent on the concentration of extracellular albumin and on the presence of exogenous fatty acids. Fatty acid-free albumin caused a concentration-dependent increase in PAF synthesis up to 5% albumin concentrations (w/v) where the amount of PAF produced was three- to four-fold higher than in controls containing no albumin. The addition of free fatty acids, particularly arachidonic acid and palmitic acid, to 5% fatty acid-free albumin media caused a concentration-dependent decrease in PAF synthesis. A 50% inhibition of PAF synthesis was observed at an arachidonic acid concentration of 120 microM and at a palmitic acid concentration of 100 microM. The inhibition of PAF production by palmitic acid was also dependent on the concentration of extracellular albumin. In 0.5% fatty acid-free albumin media, a palmitic acid concentration of 40 microM produced a 50% inhibition in PAF synthesis. The addition of palmitic acid did not affect the release of endogenous arachidonic acid during stimulation. In contrast, the addition of stearic acid up to 120 microM in 5% fatty acid-free albumin media had no effect on PAF production. The different inhibitory effects of palmitic acid and stearic acid on PAF production may be related to differences in intracellular utilization of these two fatty acids during cell stimulation.

GSK812397 is a potent entry inhibitor of X4-tropic strains of HIV-1, as demonstrated in multiple in vitro cellular assays (e.g., in peripheral blood mononuclear cells [PBMCs] and a viral human osteosarcoma [HOS] assay, mean 50% inhibitory concentrations [IC 50 s] ± standard errors of the means were 4.60 ± 1.23 nM and 1.50 ± 0.21 nM, respectively). The primary in vitro potency of GSK812397 was not significantly altered by the addition of serum proteins (2.55 [±0.12]-fold shift in the presence of human serum albumin and α-acid glycoprotein in the PBMC assay). Pharmacological characterization of GSK812397 in cell-based functional assays revealed it to be a noncompetitive antagonist of the CXCR4 receptor, with GSK812397 producing a concentration-dependent decrease in both an SDF-1-mediated chemotaxis and intracellular calcium release (IC 50 s were 0.34 ± 0.01 nM and 2.41 ± 0.50 nM, respectively). With respect to the antiviral activity of GSK812397, it was effective against a broad range of X4- and X4R5-utilizing clinical isolates. The potency and efficacy of GSK812397 were dependent on the individual isolate, with complete inhibition of infection observed with 24 of 30 isolates. GSK812397 did not show any detectable in vitro cytotoxicity and was highly selective for CXCR4, as determined using a wide range of receptors, enzymes, and transporters. Moreover, GSK812397 demonstrated acceptable pharmacokinetic properties and bioavailability across species. The data demonstrate that GSK812397 has antiviral activity against a broad range of X4-utilizing strains of HIV-1 via a noncompetitive antagonism of the CXCR4 receptor.

A series of novel N-alkoxy-arylsulfonamide HIV protease inhibitors with low picomolar enzyme activity and single digit nanomolar antiviral activity is disclosed.

Negative ion electrospray ionization, fast-atom bombardment, and low energy tandem mass spectrometry were used for the analysis of dihydroxy-eicosatrienoic acids, which contain a vicinol diol and three nonconjugated double bonds, dihydroxy-eicosatetraenoic acids, which contain a conjugated triene structure, and trihydroxy-eicosatetraenoic acids, which contain a vicinol diol and a conjugated tetraene structure. In general, the product ion spectra were qualitatively similar for both modes of ionization, but electrospray ionization was strikingly more efficient in generation of abundant carboxylate anions that could be collisionally activated to yield product ion spectra. Collision-induced dissociation fragmentation mechanisms were described generally by α-hydroxy fragmentations directed by relative positions of double bonds and were consistent with stable isotope labeling studies. Rearrangement of the conjugated triene system in dihydroxy-eicosatetraenoic acids may be described by formation of a cyclohexadiene structure. Fragmentations that involve a two-proton transfer were described best by intramolecular oxidation of a hydroxy substituent to an enolate that resulted in an extended conjugated system. Collision-induced dissociation spectra obtained for the polyhydroxy unsaturated fatty acids, which are isobaric within each class, were uniquely descriptive of individual structures.

A method has been developed for the analysis of derivatized diradylglycerols obtained from glycerophosphocholine (GPC) of transformed murine bone marrow-derived mast cells that provided high performance liquid chromatography (HPLC) separation of GPC subclasses and molecular species separation with on-line quantitation using UV detection. In addition, the derivatized diradylglycerol species were unequivocably identified by continuous flow fast-atom bombardment mass spectrometry. GPC was initially isolated by thin-layer chromatography (TLC), the phosphocholine group was hydrolyzed, and the resultant diradylglycerol was derivatized with 7-[(chlorocarbonyl)-methoxy]-4-methylcoumarin (CMMC). After separation of the derivatized subclasses by normal phase HPLC, the individual molecular species of the alkylacyl and diacyl subclasses were quantitated and collected during a subsequent reverse phase HPLC step. With an extinction coefficient of 14,700 l mol-1 cm-1 at a wavelength detection of 320 nm, the CMMC derivatives afforded sensitive UV detection (100 pmol) and quantitation of the molecular species. Continuous flow fast-atom bombardment mass spectrometry of the alkylacyl CMMC derivatives yielded abundant [MH]+ ions and a single fragment ion formed by loss of alkylketene from the sn-2 acyl group, [MH-(R = C = O)]+. No fragmentation of the sn-1 alkyl chain was observed. Diacyl derivatives also produced abundant [MH]+ ions plus two fragment ions arising from loss of RCOOH from each of the acyl substituents and two fragment ions from the loss of alkyketene from each acyl group. Individual molecular species substituents were assigned from these ions.

Par photolyse a 254 nm du t-butylhydroperoxyde en milieu aqueux contenant de la glycine, on obtient du N-acetyl ethylcarbamoyl glycinate

Electron ionization (EI) gas chromatography/mass spectrometry (GC/MS) analysis of pentafluorobenzyl ester-trimethyl sllyl ether (PFB-TMS) derivatives of hydroxy-subshtuted fatty acids provides structural information comparable to that obtained in analysis of methyl ester-trimethyl silyl ether (Me-TMS) derivatives. Use of this derivative eliminates the need to prepare two separate derivatives, the PFB-TMS derivative for molecular weight determination by electron capture ionization (negative ions) analysis and the Me-TMS derivative for structural determination by EI GC/MS analysis. The relative abundance of fragment ions observed during EI GC/MS analysis of these derivatized unsaturated fatty acids indicates the location of the —OTMS substituents relative to double bond positions in those cases studied. The most abundant fragment ions are observed when the compound contains an unsaturation two carbon atoms removed from the —OTMS ether carbon (the β-OTMS position). The “saddle effect” observed in the GC/MS analyses of some derivatized monohy— droxy unsaturated fatty acids is suggested to be due to a thermally allowed pericyclic double bond rearrangement and indicates the presence of a conjugated diene one carbon atom removed from the —OTMS ether carbon (the α-OTMS position). The saddle effect is most prominent for fatty acids that contain additional unsaturation separated by a single methylene unit from the conjugated diene moiety.