The hypothesis in the current study is that the simultaneous direct in vivo testing of thousands to millions of systematically arranged mixture-based libraries will facilitate the identification of enhanced individual compounds. Individual compounds identified from such libraries may have increased specificity and decreased side effects early in the discovery phase. Testing began by screening ten diverse scaffolds as single mixtures (ranging from 17 340 to 4 879 681 compounds) for analgesia directly in the mouse tail withdrawal model. The “all X” mixture representing the library TPI-1954 was found to produce significant antinociception and lacked respiratory depression and hyperlocomotor effects using the Comprehensive Laboratory Animal Monitoring System (CLAMS). The TPI-1954 library is a pyrrolidine bis-piperazine and totals 738 192 compounds. This library has 26 functionalities at the first three positions of diversity made up of 28 392 compounds each (26 × 26 × 42) and 42 functionalities at the fourth made up of 19 915 compounds each (26 × 26 × 26). The 120 resulting mixtures representing each of the variable four positions were screened directly in vivo in the mouse 55 °C warm-water tail-withdrawal assay (ip administration). The 120 samples were then ranked in terms of their antinociceptive activity. The synthesis of 54 individual compounds was then carried out. Nine of the individual compounds produced dose-dependent antinociception equivalent to morphine. In practical terms what this means is that one would not expect multiexponential increases in activity as we move from the all-X mixture, to the positional scanning libraries, to the individual compounds. Actually because of the systematic formatting one would typically anticipate steady increases in activity as the complexity of the mixtures is reduced. This is in fact what we see in the current study. One of the final individual compounds identified, TPI 2213-17, lacked significant respiratory depression, locomotor impairment, or sedation. Our results represent an example of this unique approach for screening large mixture-based libraries directly in vivo to rapidly identify individual compounds.Keywords: analgesia; in vivo high-throughput screening; mixture-based combinatorial libraries; opioid

•α-hydroxy-β-phenylalanine containing endomorphin analogues are reported.•Multiple analogues display high affinity for μ and δ opioid receptors.•Compound 26 produces antinociception equivalent to morphine without locomotor activity.A novel series of endomorphin-1 (EM-1) and endomorphin-2 (EM-2) analogues was synthesized, incorporating chiral α-hydroxy-β-phenylalanine (AHPBA), and/or Dmt1-Tic2 at different positions. Pharmacological activity and metabolic stability of the series was assessed. Consistent with earlier studies of β-amino acid substitution into endomorphins, multiple analogues incorporation AHPBA displayed high affinity for μ and δ opioid receptors (MOR and DOR, respectively) in radioligand competition binding assays, and an increased stability in rat brain membrane homogenates, notably Dmt-Tic-(2R,3S)AHPBA-Phe-NH2 (compound 26). Intracerebroventricular (i.c.v.) administration of 26 produced antinociception (ED50 value (and 95% confidence interval) = 1.98 (0.79–4.15) nmol, i.c.v.) in the mouse 55 °C warm-water tail-withdrawal assay, equivalent to morphine (2.35 (1.13–5.03) nmol, i.c.v.), but demonstrated DOR-selective antagonism in addition to non-selective opioid agonism. The antinociception of 26 was without locomotor activity or acute antinociceptive tolerance. This novel class of peptides adds to the potentially therapeutically relevant collection of previously reported EM analogues.

A novel and traceless strategy has been devised that allows a coupling of thioacids and dithiocarbamate-terminal amines. This strategy had been assumed to be dependent on the attachment of a functional equivalent of a cysteine side chain in earlier native chemical ligation approaches. This approach enables the traceless removal of CS2 to directly generate the desired amide bond and is compatible with a range of unprotected side chains of amino acid. The ability to produce amide or peptides by a traceless removal of the auxiliary is a significant virtue of the method. Meanwhile, the application of this new peptide-bond-forming reaction to the synthesis of novel endomorphin (EM) derivatives with various binding potencies was realized.

A positional scanning cyclic peptide library was generated using a penta-peptide thioester scaffold. Glycine was fixed at position R1. Diaminopropionic acid was fixed at position R3, with its γ-amino attaching to an anthraniloyl group. Positions R2 and R4 contained 36 l- and d- amino acids and position R5 contained 19 l- amino acids. Cyclization was performed in a mixture of acetonitrile and 1.5 M aqueous imidazole solution (7:1 v/v) at room temperature for 5 days. No significant cross-oligomerization was detected under the cyclization conditions. The library was screened in a binding assay for mu opioid receptor, identifying the active amino acid mixture at each position. A total of 40 individual cyclic peptides were identified and synthesized by the combinations of the most active amino acid mixtures found at three positions 5 × 4 × 2. Two cyclic peptides exhibited high binding affinities to opioid receptor. The most active cyclic peptide in the library was yielded to have Tyr at R2, d-Lys at R4, and Tyr at R5. Further investigation on this compound revealed the side chain-to-tail isomer to have greater binding affinity (14 nM) than the head-to-tail isomer (39 nM). Both isomers were selective for the mu-opioid receptor.Keywords: cyclic peptide; fluorescent label; mu selective ligand; opioid ligand; positional scanning library; synthetic cyclic peptide library

The use of the harmonic mean model for predicting the activities of a given mixture and its constituents has not previously been explored in the context of combinatorial libraries and drug discovery. Herein, the analyses of historical data confirm the harmonic mean as an accurate predictor of mixture activity. The implications of these results are discussed.Keywords: combinatorial libraries; drug discovery; harmonic mean model; mixture activity

A facile approach to the synthesis of 2,3,6-trisubstituted-5,6-dihydroimidazo[2,1-b]thiazole was reported. A resin bound cyclic thiourea was formed by the treatment of a resin bound diamine with 1,1′-thiocarbonyldiimidazole, and then reacted with a α-haloketone to generate a resin bound isothiourea. HF treatment of the resin bound isothiourea resulted in the cleavage of the product and simultaneous formation of an enamine bond. This led to the formation of the 2,3,6-trisubstituted-5,6-dihydroimidazo[2,1-b]thiazole in high yield and purity.A facile approach to the synthesis of 2,3,6-trisubstituted-5,6-dihydroimidazo[2,1-b]thiazole derivatives was reported.

We have developed an efficient solid-phase synthetic approach for the synthesis of 2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine derivatives. The methodology is of value for high throughput synthesis of these potentially bioactive molecules.

The macrolactonization of peptide thioester to yield cyclic depsipeptides was developed using imidazole as a catalyst. This strategy was applied to the synthesis of kahalalide B and its analogues.

A facile solid-phase synthetic approach to the synthesis of 3,4-disubstituted-2-aminothiazolium derivatives was reported. Functionalized aminomethylphenyl silica gel was used as a ‘volatilizable’ support. The products were cleaved with 10% HF and were obtained in high yields and purities.A facile solid-phase synthetic approach to the synthesis of 3,4-disubstituted-2-aminothiazole derivatives was reported. Functionalized aminomethylphenyl silica gel was used as a ‘volatilizable’ support. The products were cleaved with 10% HF and were obtained in high yields and purities.

Synthetic combinatorial methods now make it practical to readily produce hundreds of thousands of individual compounds, but it is clearly impractical to screen each separately in vivo. We theorized that the direct in vivo testing of mixture-based combinatorial libraries during the discovery phase would enable the identification of novel individual compounds with desirable antinociceptive profiles while simultaneously eliminating many compounds with poor absorption, distribution, metabolism, or pharmacokinetic properties. The TPI 1346 small-molecule combinatorial library is grouped in 120 mixtures derived from 26 functionalities at the first three positions and 42 functionalities at the fourth position of a pyrrolidine bis-cyclic guanidine core scaffold, totaling 738,192 compounds. These 120 mixtures were screened in vivo using the mouse 55°C warm water tail-withdrawal assay to identify mixtures producing antinociception. From these data, two fully defined individual compounds (TPI 1818-101 and TPI 1818-109) were synthesized. These were examined for antinociceptive, respiratory, locomotor, and conditioned place preference effects. The tail-withdrawal assay consistently demonstrated distinctly active mixtures with analgesic activity that was blocked by pretreatment with the non-selective opioid antagonist, naloxone. Based on these results, synthesis and testing of TPI 1818-101 and 1818-109 demonstrated a dose-dependent antinociceptive effect three to five times greater than morphine that was antagonized by mu- or mu- and kappa-opioid receptor selective antagonists, respectively. Neither 1818-101 nor 1818-109 produced significant respiratory depression, hyperlocomotion, or conditioned place preference. Large, highly diverse mixture-based libraries can be screened directly in vivo to identify individual compounds, potentially accelerating the development of promising therapeutics.

A promising method for the synthesis of cyclic peptides through the direct aminolysis of peptide thioesters is presented. The cyclization step was carried out in a mixture of acetonitrile and 1.5 M aqueous imidazole solution with no observable oligomers. Studies on the N- and C-terminal residues show that the choice of C-terminal residue has a more significant effect on the success rate of cyclization than the choice at the N-terminal residue.

A promising method for the high-throughput synthesis of linear C-hydroxyalkylamido peptidomimetics and β-turn cyclic peptidomimetics via “volatilizable” aminoalkyl functionalized silica gels is presented. Boc amino acids and carboxylic acids were coupled on functionalized aminoalkyl silica gels using a standard DIC/HOBt coupling protocol. After peptide synthesis, the resin bound peptide was cleaved using a two-step process to obtain the linear C-hydroxyalkylamido peptidomimetics. β-Turn cyclic peptidomimetics were generated by intramolecular SNAr cyclization in an aqueous solution. Both the linear and the cyclic peptidomimetics were obtained with good to excellent yields and purities through a “one-pot” reaction.

A novel and traceless strategy has been devised that allows a coupling of thioacids and dithiocarbamate-terminal amines. This strategy had been assumed to be dependent on the attachment of a functional equivalent of a cysteine side chain in earlier native chemical ligation approaches. This approach enables the traceless removal of CS2 to directly generate the desired amide bond and is compatible with a range of unprotected side chains of amino acid. The ability to produce amide or peptides by a traceless removal of the auxiliary is a significant virtue of the method. Meanwhile, the application of this new peptide-bond-forming reaction to the synthesis of novel endomorphin (EM) derivatives with various binding potencies was realized.