Synthesis and Pharmacological Evaluation of Enantiopure N-Substituted Ortho-c Oxide-Bridged 5-Phenylmorphans.

The design of enantiopure stereoisomers of N-2-phenylcyclopropylmethyl-substituted ortho-c oxide-bridged phenylmorphans, the E and Z isomers of an N-cinnamyl moiety, and N-propyl enantiomers were based on combining the most potent oxide-bridged phenylmorphan (the ortho-c isomer) with the most potent N-substituent that we previously found with a 5-(3-hydroxy)phenylmorphan (i.e., N-2-phenylcyclopropyl methyl moieties, N-cinnamyl, and N-propyl substituents). The synthesis of the eight enantiopure N-2-phenylcyclopropylmethyl ortho-c oxide-bridged phenylmorphans and six additional enantiomers of the N-substituted ortho-c oxide-bridged phenylmorphans (N-E and Z-cinnamyl compounds, and N-propyl compounds) was accomplished. The synthesis started from common intermediates (3R,6aS,11aS)-10-methoxy-1,3,4,5,6,11a-hexahydro-2H-3,6a-methano-benzofuro[2,3-c]azocine (+)-6 and its enantiomer, (3S, 6aR, 11aR)-(-)-6, respectively. The enantiomers of ±-6 were obtained through salt formation with (S)-(+)- and (R)-(-)-p-methylmandelic acid, and the absolute configuration of the (R)-(-)-p-methylmandelate salt of (3S, 6aR, 11aR)-(-)-6 was determined by single-crystal X-ray analysis. The enantiomeric secondary amines were reacted with N-(2-phenylcyclopropyl)methyl derivatives, 2-(E)-cinnamyl bromide, and (Z)-3-phenylacrylic acid. These products led to all of the desired N-derivatives of the ortho-c oxide-bridged phenylmorphans. Their opioid receptor binding affinity was measured. The compounds with MOR affinity < 50 nM were examined for their functional activity in the forskolin-induced cAMP accumulation assay. Only the enantiomer of the N-phenethyl ortho-c oxide-bridged phenylmorphan ((-)-1), and only the (3S,6aR,11aR)-2-(((1S,2S)-2-phenylcyclopropyl)methyl)-1,3,4,5,6,11a-hexahydro-2H-3,6a-methanobenzofuro[2,3-c]azocin-10-ol isomer ((+)-17), and the N-phenylpropyl derivative ((-)-25) had opioid binding affinity < 50 nM. Both (-)-1 and (-)-25 were partial agonists in the cAMP assay, with the former showing high potency and low efficacy, and the latter with lower potency and less efficacy. Most interesting was the N-2-phenylcyclopropylmethyl (3S,6aR,11aR)-2-(1S,2S)-enantiomer ((+)-17). That compound had good MOR binding affinity (Ki = 11.9 nM) and was found to have naltrexone-like potency as a MOR antagonist (IC50 = 6.92 nM).

Design, Synthesis, and In Vivo Evaluation of C1-Linked 4,5-Epoxymorphinan Haptens for Heroin Vaccines.

In our continuing effort to develop effective anti-heroin vaccines as potential medications for the treatment of opioid use disorder, herein we present the design and synthesis of the haptens: 1-AmidoMorHap (1), 1-AmidoMorHap epimer (2), 1 Amido-DihydroMorHap (3), and 1 Amido-DihydroMorHap epimer (4). This is the first report of hydrolytically stable haptenic surrogates of heroin with the attachment site at the C1 position in the 4,5-epoxymorophinan nucleus. We prepared respective tetanus toxoid (TT)-hapten conjugates as heroin vaccine immunogens and evaluated their efficacy in vivo. We showed that all TT-hapten conjugates induced high antibody endpoint titers against the targets but only haptens 2 and 3 can induce protective effects against heroin in vivo. The epimeric analogues of these haptens, 1 and 4, failed to protect mice from the effects of heroin. We also showed that the in vivo efficacy is consistent with the results of the in vitro drug sequestration assay. Attachment of the linker at the C1 position induced antibodies with weak binding to the target drugs. Only TT-2 and TT-3 yielded antibodies that bound heroin and 6-acetyl morphine. None of the TT-hapten conjugates induced antibodies that cross-reacted with morphine, methadone, naloxone, or naltrexone, and only TT-3 interacted weakly with buprenorphine, and that subtle structural difference, especially at the C6 position, can vastly alter the specificity of the induced antibodies. This study is an important contribution in the field of vaccine development against small-molecule targets, providing proof that the chirality at C6 in these epoxymorphinans is a vital key to their effectiveness.

X-ray Crystallography and Unexpected Chiroptical Properties Reassign the Configuration of Haliclonadiamine.

Haliclonadiamine and papuamine are bis-indane marine natural products isolated from the marine sponge Haliclona sp. Their relative structures were previously reported to differ by inversion at only one of their eight shared stereocenters. Here X-ray crystallography shows the opposite to be true: papuamine has a 1R,3S,8R,9S,14S,15R,20S,22R configuration, while haliclonadiamine has a 1S,3R,8S,9R,14R,15S,20R,22R configuration. Paradoxically the ECD of each structure displays a negative Cotton effect. X-ray crystallography reveals the two structures adopt similar conformations of their 13-membered macrocyclic core that comprises a configurationally relevant diene. B97x-D/Def2-TZVPP-(MeOH)-calculated ECD supports the diene configuration with the macrocycle dominating the ECD Cotton effect for haliclonadiamine and papuamine. Additional crystallographic and chiroptical analyses of three sponge samples from geographically distant locations indicate this pair of natural products always exists as a configurationally related couple. The co-discovery of a biosynthetic precursor, halichondriamine C, present in these same Haliclona samples must be considered when discussing any biosynthetic pathway. Taken together, this work justifies a reassignment of haliclonadiamine's structure and opens the question of how this complex stereochemical relationship between haliclonadiamine and palauamine arises biosynthetically.

Reaction Kinetics Direct a Rational Synthesis of an HIV-1 Inactivator of Nucleocapsid Protein 7 and Provide Mechanistic Insight into Cellular Metabolism and Antiviral Activity.

Mercaptobenzamide thioester SAMT-247 is a non-toxic, mutation-resistant HIV-1 maturation inhibitor with a unique mechanism of antiviral activity. NMR spectroscopic analyses of model reactions that mimic the cellular environment answered fundamental questions about the antiviral mechanism and inspired a high-yielding (64 % overall), scalable (75 mmol), and cost-effective ($4 mmol-1 ) three-step synthesis that will enable additional preclinical evaluation.

Total Synthesis of Sarpagine-Related Bioactive Indole Alkaloids.

Extension of the asymmetric Pictet-Spengler reaction to bulkier Nb -alkylated tryptophan derivatives resulted in an improved stereospecific access to the key bicyclo[3.3.1]nonane core of bioactive C-19 methyl substituted sarpagine/macroline/ajmaline indole alkaloids with excellent diastereoselectivity by internal asymmetric induction. Complete stereocontrol of the C-19 methyl function in either the α- or ß-configuration was achieved, which enables the total synthesis of any member from this group of thirty alkaloids. We report herein, the total synthesis of macrocarpines (A-C) 1-3, talcarpine 4, N(4)-methyl-N(4),21-secotalpinine 5, dihydroperaksine 8 and deoxyperaksine 9.

A High Aspect Ratio Bifurcated 128-Microchannel Microfluidic Device for Environmental Monitoring of Explosives.

Design and evolution of explosives monitoring and detection platforms to address the challenges of trace level chemical identification have led investigations into the use of intricately designed microfluidic devices. Microfluidic devices are unique tools that possess distinct characteristics that, when designed properly and configured with optical and fluidic components, can produce detection platforms with unmatched performance levels. Herein, we report the design, fabrication and integration of a bifurcated high aspect ratio microfluidic device containing 128 microchannels (40 mm × 40 µm × 250 µm; L × W × H) for explosives detection at trace levels. Aspect ratios measuring >6:1 support improved receptor-target molecule interactions, higher throughput and extremely low limits of detection (LOD). In addition to superior assay sensitivity, the bifurcated microfluidic device provides greater durability and versatility for substrate modification. Using the explosive 2,4,6-trinitrotoluene (TNT) as the model compound in a fluorescence-based displacement immunoassay, we report LODs for TNT at 10 parts-per-trillion (pptr) using a neutravidin-coated biotinylated anti-TNT microfluidic device. Solution to wall interactions were also simulated in COMSOL Multiphysics to understand fluid flow characteristics. Reynolds numbers were calculated to be 0.27⁻2.45 with a maximum pressure of 1.2 × 10-2 psi.

Engineering Immunological Tolerance Using Quantum Dots to Tune the Density of Self-Antigen Display.

Treatments for autoimmunity - diseases where the immune system mistakenly attacks self-molecules - are not curative and leave patients immunocompromised. New studies aimed at more specific treatments reveal development of inflammation or tolerance is influenced by the form self-antigens are presented. Using a mouse model of multiple sclerosis (MS), we show for the first time that quantum dots (QDs) can be used to generate immunological tolerance by controlling the density of self-antigen on QDs. These assemblies display dense arrangements of myelin self-peptide associated with disease in MS, are uniform in size (<20 nm), and allow direct visualization in immune tissues. Peptide-QDs rapidly concentrate in draining lymph nodes, co-localizing with macrophages expressing scavenger receptors involved in tolerance. Treatment with peptide-QDs reduces disease incidence 10-fold. Strikingly, the degree of tolerance - and the underlying expansion of regulatory T cells - correlates with the density of myelin molecules presented on QDs. A key discovery is that higher numbers of tolerogenic particles displaying lower levels of self-peptide are more effective for inducing tolerance than fewer particles each displaying higher densities of peptide. QDs conjugated with self-antigens could serve as a new platform to induce tolerance, while visualizing QD therapeutics in tolerogenic tissue domains.

Concise Total Synthesis of (-)-Affinisine Oxindole, (+)-Isoalstonisine, (+)-Alstofoline, (-)-Macrogentine, (+)-Na -Demethylalstonisine, (-)-Alstonoxine†A, and (+)-Alstonisine.

A highly enantio- and diastereoselective strategy to access any member of the sarpagine/macroline family of oxindole alkaloids via internal asymmetric induction was developed from readily available d-(+)-tryptophan. At the center of this approach was the diastereospecific generation of the spiro[pyrrolidine-3,3'-oxindole] moiety at an early stage via a tert-butyl hypochlorite-promoted oxidative rearrangement of a chiral tetrahydro-ß-carboline derivative. This key branching point determined the spatial configuration at the C-7 spiro center to be entirely 7R or 7S. Other key stereospecific processes were the asymmetric Pictet-Spengler reaction and Dieckmann cyclization, which were scalable to the 600 and 150 gram levels, respectively. Execution of this approach resulted in first enantiospecific total synthesis of (+)-isoalstonisine and (-)-macrogentine from the chitosenine series (7R), as well as (+)-alstonisine, (+)-alstofoline, (-)-alstonoxine A and (+)-Na -demethylalstonisine from the alstonisine series (7S).

Iodobenzene-Catalyzed Synthesis of Phenanthridinones via Oxidative C-H Amidation.

We report a novel synthesis of phenanthridinones from N-methoxybenzamides using an oxidative C-H amidation reaction at room temperature in open air with modest to excellent yields. This method demonstrated unprecedented substrate scope. In particular, it solved the long-standing challenge in the synthesis of phenanthridinones with sterically demanding substitutions.

One-pot sequential reaction to 2-substituted-phenanthridinones from N-methoxybenzamides.

The sequential use of a hypervalent iodine reagent leads to the one-pot synthesis of 2-bromo/chloro-phenanthridinones via an amidation of arenes followed by a regioselective halogenation reaction. These consecutive C-H functionalization reactions can be used efficiently to construct 2-substituted-phenanthridinones at room temperature with good to high yields. Application of the current method is highlighted by the concise synthesis of the natural product PJ34.

Modulation of opioid receptor affinity and efficacy via N-substitution of 9ß-hydroxy-5-(3-hydroxyphenyl)morphan: Synthesis and computer simulation study.

The enantiomers of a variety of N-alkyl-, N-aralkyl-, and N-cyclopropylalkyl-9ß-hydroxy-5-(3-hydroxyphenyl)morphans were synthesized employing cyanogen bromide and K2CO3 to improve the original N-demethylation procedure. Their binding affinity to the µ-, δ-, and κ-opioid receptors (ORs) was determined and functional (GTPγ35S) assays were carried out on those with reasonable affinity. The 1R,5R,9S-enantiomers (1R,5R,9S)-(-)-5-(3-hydroxyphenyl)-2-(4-nitrophenethyl)-2-azabicyclo[3.3.1]nonan-9-ol (1R,5R,9S-16), (1R,5R,9S)-(-) 2-cinnamyl-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]nonan-9-ol (1R,5R,9S-20), and (1R,5R,9S)-(-)-5-(3-hydroxyphenyl)-2-(4-(trifluoromethyl)phenethyl)-2-azabicyclo[3.3.1]nonan-9-ol (1R,5R,9S-15), had high affinity for the µ-opioid receptor (e.g., 1R,5R,9S-16: Ki=0.073, 0.74, and 1.99nM, respectively). The 1R,5R,9S-16 and 1R,5R,9S-15 were full, high efficacy µ-agonists (EC50=0.74 and 18.5nM, respectively) and the former was found to be a partial agonist at δ-OR and an antagonist at κ-OR, while the latter was a partial agonist at δ-OR and κ-OR in the GTPγ35S assay. The enantiomer of 1R,5R,9S-16, (+)-1S,5S,9R-16 was unusual, it had good affinity for the µ-OR (Ki=26.5nM) and was an efficacious µ-antagonist (Ke=29.1nM). Molecular dynamics simulations of the µ-OR were carried out with the 1R,5R,9S-16 µ-agonist and the previously synthesized (1R,5R,9S)-(-)-5-(9-hydroxy-5-(3-hydroxyphenyl-2-phenylethyl)-2-azabicyclo[3.3.1]nonane (1R,5R,9S-(-)-NIH 11289) to provide a structural basis for the observed high affinities and efficacies. The critical roles of both the 9ß-OH and the p-nitro group are elucidated, with the latter forming direct, persistent hydrogen bonds with residues deep in the binding cavity, and the former interacting with specific residues via highly structured water bridges.

Design, Synthesis, and Biological Evaluation of Structurally Rigid Analogues of 4-(3-Hydroxyphenyl)piperidine Opioid Receptor Antagonists.

In order to gain additional information concerning the active conformation of the N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine (1) class of opioid receptor antagonists, procedures were developed for the synthesis of structurally rigid N-substituted-6-(3-hydroxyphenyl)3-azabicyclo[3.1.0]hexane and 3-methyl-4-(3-hydroxyphenyl)-4-azabicyclo[4.1.0]heptanes. Evaluation of the conformationally constrained series in a [35S]GTPγS assay showed that structural rigid compounds having the 3-hydroxyphenyl group locked in the piperidine equatorial orientation had potencies equal to or better than similar compounds having more flexible structures similar to 1. The studies of the rigid compounds also suggested that the 3-methyl group present in compound 1 type antagonists may not be necessary for their pure opioid antagonist properties.

Efficacy, but not antibody titer or affinity, of a heroin hapten conjugate vaccine correlates with increasing hapten densities on tetanus toxoid, but not on CRM197 carriers.

Vaccines against drugs of abuse have induced antibodies in animals that blocked the biological effects of the drug by sequestering the drug in the blood and preventing it from crossing the blood-brain barrier. Drugs of abuse are too small to induce antibodies and, therefore, require conjugation of drug hapten analogs to a carrier protein. The efficacy of these conjugate vaccines depends on several factors including hapten design, coupling strategy, hapten density, carrier protein selection, and vaccine adjuvant. Previously, we have shown that 1 (MorHap), a heroin/morphine hapten, conjugated to tetanus toxoid (TT) and mixed with liposomes containing monophosphoryl lipid A [L(MPLA)] as adjuvant, partially blocked the antinociceptive effects of heroin in mice. Herein, we extended those findings, demonstrating greatly improved vaccine induced antinociceptive effects up to 3% mean maximal potential effect (%MPE). This was obtained by evaluating the effects of vaccine efficacy of hapten 1 vaccine conjugates with varying hapten densities using two different commonly used carrier proteins, TT and cross-reactive material 197 (CRM197). Immunization of mice with these conjugates mixed with L(MPLA) induced very high anti-1 IgG peak levels of 400-1500 µg/mL that bound to both heroin and its metabolites, 6-acetylmorphine and morphine. Except for the lowest hapten density for each carrier, the antibody titers and affinity were independent of hapten density. The TT carrier based vaccines induced long-lived inhibition of heroin-induced antinociception that correlated with increasing hapten density. The best formulation contained TT with the highest hapten density of ≥30 haptens/TT molecule and induced %MPE of approximately 3% after heroin challenge. In contrast, the best formulation using CRM197 was with intermediate 1 densities (10-15 haptens/CRM197 molecule), but the %MPE was approximately 13%. In addition, the chemical synthesis of 1, the optimization of the conjugation method, and the methods for the accurate quantification of hapten density are described.

Synthesis of aza and carbocyclic ß-carbolines for the treatment of alcohol abuse. Regiospecific solution to the problem of 3,6-disubstituted ß- and aza-ß-carboline specificity.

A novel two step protocol was developed to gain regiospecific access to 3-substituted ß- and aza-ß-carbolines, 3-PBC (1), 3-ISOPBC (2), ßCCt (3), 6-aza-3-PBC (4) and 6-aza-3-ISOPBC (5). These ß-carbolines (1-3) are potential clinical agents to reduce alcohol self-administration, especially 3-ISOPBC·HCl (2·HCl) which appears to be a potent anti-alcohol agent active against binge drinking in a rat model of maternally deprived (MD) rats. The method consists of two consecutive palladium-catalyzed reactions: a Buchwald-Hartwig amination followed by an intramolecular Heck-type cyclization in high yield.

PABA/NO lead optimization: Improved targeting of cytotoxicity to glutathione S-transferase P1-overexpressing cancer cells.

PABA/NO [O(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino) diazen-1-ium-1,2-diolate] is a nitric oxide (NO)-releasing arylating agent designed to be selectively activated by reaction with glutathione (GSH) on catalysis by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancer cells. PABA/NO has proven active in several cancer models in vitro and in vivo, but its tendency to be metabolized via a variety of pathways, some that generate inactive metabolites and hydrolysis products, limits its potential as a drug. Here we show that a simple replacement of cyano for nitro at the 4 position to give compound 4b ('p-cyano-PABA/NO') has the dual effect of slowing the undesired side reactions while enhancing the proportion of NO release and arylating activity on catalysis by GSTP1. Compound 4b showed increased resistance to hydrolysis and uncatalyzed reaction with GSH, along with a more favorable product distribution in the presence of GSTP1. It also showed significant proapoptotic activity. The data suggest p-cyano-PABA/NO to be a more promising prodrug than PABA/NO, with better selectivity toward cancer cells.

Using click chemistry toward novel 1,2,3-triazole-linked dopamine D3 receptor ligands.

The dopamine D3 receptor (D3R) is a target of pharmacotherapeutic interest in a variety of neurological disorders including schizophrenia, Parkinson's disease, restless leg syndrome, and drug addiction. A common molecular template used in the development of D3R-selective antagonists and partial agonists incorporates a butylamide linker between two pharmacophores, a phenylpiperazine moiety and an extended aryl ring system. The series of compounds described herein incorporates a change to that chemical template, replacing the amide functional group in the linker chain with a 1,2,3-triazole group. Although the amide linker in the 4-phenylpiperazine class of D3R ligands has been previously deemed critical for high D3R affinity and selectivity, the 1,2,3-triazole moiety serves as a suitable bioisosteric replacement and maintains desired D3R-binding functionality of the compounds. Additionally, using mouse liver microsomes to evaluate CYP450-mediated phase I metabolism, we determined that novel 1,2,3-triazole-containing compounds modestly improves metabolic stability compared to amide-containing analogues. The 1,2,3-triazole moiety allows for the modular attachment of chemical subunit libraries using copper-catalyzed azide-alkyne cycloaddition click chemistry, increasing the range of chemical entities that can be designed, synthesized, and developed toward D3R-selective therapeutic agents.

Chiral resolution and absolute configuration of the enantiomers of the psychoactive "designer drug" 3,4-methylenedioxypyrovalerone.

Illicit rac-MDPV (3,4-methylenedioxypyrovalerone), manufactured in clandestine labs, has become widely abused for its cocaine-like stimulant properties. It has recently been found as one of the toxic materials in the so-called "bath salts," producing, among other effects, psychosis and tachycardia in humans when introduced by any of the several routes of administration (e.g., intravenous, oral, etc.). The considerable toxicity of this "designer drug" probably resides in one of the enantiomers of the racemate. In order to obtain a sufficient amount of the enantiomers of rac-MDPV to determine their activity, we improved the known synthesis of rac-MDPV and found chemical resolving agents, (+)- and (-)-2'-bromotetranilic acid, that gave the MDPV enantiomers in >96% enantiomeric excess as determined by (1) H nuclear magnetic resonance and chiral high-performance liquid chromatography. The absolute stereochemistry of these enantiomers was determined by single-crystal X-ray diffraction studies.

Synthesis of 1,2-dihydro-2-oxo-4-quinolinyl phosphates from 2-acyl-benzoic acids.

We report a facile synthesis of 1,2-dihydro-2-oxo-4-quinolinyl phosphates (1a-l) starting from 2-acyl-benzoic acids (2a-l) in the presence of phosphoryl azides via a one-pot cascade reaction involving a Curtius rearrangement, an intramolecular nucleophilic addition of the enol carbon to the isocyanate intermediate, and an addition-elimination of the enol oxygen to the phosphoryl azide. During the reaction three new bonds are formed under mild conditions to yield 1,2-dihydro-2-oxo-4-quinolinyl phosphates in modest yields.

General strategy for synthesis of C-19 methyl-substituted sarpagine/macroline/ajmaline indole alkaloids including total synthesis of 19(S),20(R)-dihydroperaksine, 19(S),20(R)-dihydroperaksine-17-al, and peraksine.

A detailed account of the development of a general strategy for synthesis of the C-19 methyl-substituted alkaloids including total synthesis of 19(S),20(R)-dihydroperaksine-17-al (1), 19(S),20(R)-dihydroperaksine (2), and peraksine (6) is presented. Efforts directed toward the total synthesis of macrosalhine chloride (5) are also reported. Important to success is the sequence of chemical reactions which include a critical haloboration reaction, regioselective hydroboration, and controlled oxidation (to provide sensitive enolizable aldehydes at C-20). In addition, the all-important Pd-catalyzed α-vinylation reaction has been extended to a chiral C-19 alkyl-substituted substrate for the first time. Synthesis of the advanced intermediate 64 completes an improved formal total synthesis of talcarpine (26) and provides a starting point for synthesis of macroline-related alkaloids 27-31. Similarly, extension of this synthetic strategy in the ring A oxygenated series should provide easy access to the northern hemisphere 32b of the bisindoles angustricraline, alstocraline, and foliacraline (Figure 4 ).

Synthesis of enantiopure 10-nornaltrexones in the search for Toll-like receptor 4 antagonists and opioid ligands.

10-Nornaltrexones (3-(cyclopropylmethyl)-4a,9-dihydroxy-2,3,4,4a,5,6-hexahydro-1H-benzofuro[3,2-e]isoquinolin-7(7aH)-one, 1) have been underexploited in the search for better opioid ligands, and their enantiomers have been unexplored. The synthesis of trans-isoquinolinone 2 (4-aH, 9-O-trans-9-methoxy-3-methyl-2,3,4,4a,5,6-hexahydro-1H-benzofuro[3,2-e]isoquinolin-7(7aH)-one) was achieved through a nonchromatographic optimized synthesis of the intermediate pyridinyl compound 12. Optical resolution was carried out on 2, and each of the enantiomers were used in efficient syntheses of the "unnatural" 4aR,7aS,12bR-(+)-1) and its "natural" enantiomer (-)-1. Addition of a 14-hydroxy (the 4a-hydroxy) group in the enantiomeric isoquinolinones, (+)- and (-)-2), gave (+)- and (-)-10-nornaltrexones. A structurally unique tetracyclic enamine, (12bR)-7,9-dimethoxy-3-methyl-1,2,3,7-tetrahydro-7,12b-methanobenzo[2,3]oxocino[5,4-c]pyridine, was found as a byproduct in the syntheses and offers a different opioid-like skeleton for future study.