4-Methylideneisoxazolidin-5-ones--a new class of alpha-methylidene-gamma-lactones with high cytostatic activity

A novel, general method of synthesis of 4-methylideneisoxazolidin-5-ones 10 is described. The target compounds were synthesized starting from ethyl 2-diethoxyphosphoryl-2-alkenoates 6 or dicyclohexylammonium 4-diethoxyphosphoryl-2-alkenoates 7. Addition of N-methylhydroxylamine hydrochloride to these Michael acceptors, lactonization to 4-diethoxyphosphorylisoxazolidin-5-ones 9, and Horner-Wadsworth-Emmons olefination of formaldehyde using 9 gave the title isoxazolidinones 10. All obtained compounds were tested against L-1210, HL-60, and NALM-6 leukemia cell lines. Several isoxazolidinones 10 were found to be very potent with IC(50)<1 microM. The highest cytostatic activity against HL-60 was observed for 10a and against NALM-6 for 10b with IC(50) values of 0.74 and 0.34 microM, respectively.     

Apoptosis-mediated cytotoxic effects of parthenolide and the new synthetic analog MZ-6 on two breast cancer cell lines

The search for effective plant-derived anti-cancer agents or their synthetic analogs has continued to gain interest in drug development. The anti-cancer activity of parthenolide (PTL) isolated from Tanacetum parthenium, has been attributed to the presence of α-methylene-γ-lactone skeleton. In the present study we aimed to investigate the anti-cancer potential of a new synthetic compound, 3-isopropyl-2-methyl-4-methyleneisoxazolidin-5-one (MZ-6), with the same as in PTL α-methylene-γ-lactone motif, on two breast cancer cell lines, MCF-7 and MDA-MB-231. For comparison, PTL was included in the study. PTL and MZ-6 reduced the number of viable MCF-7 and MDA-MB-231 cells, with half maximal inhibitory concentration values between 6 and 9 μM. Both compounds dose-dependently inhibited incorporation of [³H]thymidine, up-regulated Bax and down regulated Bcl-2 mRNA. The levels of the end product of lipid peroxidation, malondialdehyde, were significantly higher. In MCF-7 cells, MZ-6 induced early apoptosis and cell cycle arrest in G0/G1 phase. The effect produced by MZ-6 was much stronger compared with PTL. In MDA-MB-231 cells, both tested compounds had similar effect and induced mostly late apoptosis. In conclusion, the observed anticancer activity makes MZ-6 an attractive drug candidate and shows that simple analogs of α-methylene-γ-lactones can be good substitutes for more complex structures isolated from plants.     

Specificity of antisera obtained to substance P and its C-terminal hexapeptide

Synthetic fragments and analogs were used to characterize specificity of antisera to SP and SP6-11. [Tyr8] SP and [Lys6] SP6-11 were both used as radioiodinated ligands. The latter was conjugated with Bolton-Hunter reagents before labelling. In both systems, the C-terminal pentapeptide SP7-11 was the shortest fragment showing antigenic identity with Substance P molecule. Substitution of different amino acid residues in SP6-11 by His or Gly showed that all but Glu6 take part in the structure of the antigenic determinant.     

Inhibitory effect of thyrotropin releasing hormone on spontaneous proliferation of mouse spleen lymphocytes in vitro.

The effects of thyrotropin releasing hormone (TRH) and of TRH-like tripeptide pGlu-His-Gly-OH (colon mitosis inhibitor, CMI) on spontaneous proliferation of murine splenocytes were investigated in vitro. The 3H-thymidine incorporation into splenocyte DNA was used as an index of proliferation. It was found that TRH suppressed the proliferation of splenocytes. In contrast, CMI was ineffective by itself but used together with TRH blocked the effect of the latter.     

Synthesis of linear and cyclic opioid‐based peptide analogs containing multiple N‐methylated amino acid residues

A series of six novel opioid peptide analogs containing one to three N‐methylamino acid residues, and six cyclic counterparts of these peptides were prepared by the solid‐phase method. Introduction of two consecutive N‐methylated amino acids, as well as cyclization of such conformationally constrained sequences, turned out to be challenging. The use of a recently reported triazine‐based coupling reagent, 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholinium toluene‐4‐sulfonate, enabled the synthesis and cyclization of the designed analogs in acceptable yields and with a lesser amount of by‐products than observed with the standard coupling reagents such as TBTU or HATU.Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Functional comparison of two evolutionary conserved insect neurokinin-like receptors

Tachykinins are multifunctional neuropeptides that have been identified in vertebrates as well as invertebrates. The C-terminal FXGXRa-motif constitutes the consensus active core region of invertebrate tachykinins. In Drosophila, two putative G protein-coupled tachykinin receptors have been cloned: DTKR and NKD. This study focuses on the functional characterization of DTKR, the Drosophila ortholog of the stable fly's tachykinin receptor (STKR). Tachykinins containing an alanine residue instead of the highly conserved glycine (FXAXRa) display partial agonism on STKR-mediated Ca(2+)-responses, but not on cAMP-responses. STKR therefore seems to differentiate between a number of tachykinins. Gly- and Ala-containing tachykinins are both encoded in the Drosophila tachykinin precursor, thus raising the question of whether DTKR can also distinguish between these two tachykinin types. DTKR was activated by all Drosophila tachykinins and inhibited by tachykinin antagonists. Ala-containing analogs did not produce the remarkable activation behavior previously observed with STKR, suggesting different mechanisms of discerning ligands and/or activating effector pathways for STKR and DTKR.     

Synthesis and biological activity of substance P C-terminal hexapeptide analogues: structure-activity studies

The synthesis of six hexapeptide analogues of C-terminal Substance P fragment containing alpha, beta-dehydrophenylalanine (delta Phe) in the position 7 or 8 is described. The effect of the structural changes on the hypotensive activity and antigenic properties of analogues was compared. It was found that substitution of delta Phe in various analogues of C-terminal hexapeptide of Substance P resulted in different effects on the hypotensive activity. The analogues [Glp6, delta Phe7]SP6-11 and [Glp6, delta Phe8]SP6-11 retained 70% and 45% of hypotensive activity of the C-terminal hexapeptide of Substance P, respectively but they showed a completely destroyed antigenic determinant. The analogues containing additionally Sar or His in the position 9 showed a complete lack of both: hypotensive activity and expression of the antigenic determinant of Substance P.     

Opioid receptors and their selective ligands

Opioid receptors (micro, delta, and kappa) belong to a large family of G protein-coupled receptors and play an important physiological role. Stimulation of these receptors triggers analgesic effects and affects the function of gastrointestinal tract. The discovery of opioid peptides, which are endogenous ligands of opioid receptors, including delta-selective enkephalins, kappa-selective dynorphins, and micro-selective endomorphins, initiated their structure-activity relationship studies. For the last 30 years, hundreds of analogs of opioid peptides have been synthesized in an effort to obtain the compounds more active, selective, and resistant to biodegradation than the endogenous ligands. Different unnatural amino acids, as well as cyclisation procedures, leading to conformationaly restricted analogs, were employed. All these modifications resulted in obtaining very selective agonists and antagonists with high affinity at micro-, dlta-, and kappa-opioid receptors, which are extremely useful tools in further studies on the pharmacology of opioid receptors in a mammalian organism.     

Chemical immobilization of free-ranging dhole (Cuon alpinus), binturong (Arctictis binturong), and yellow-throated marten (Martes flavigula) in Thailand

No published information exists on the chemical immobilization of free-ranging dholes (Cuon alpinus), binturongs (Arctictis binturong), or yellow-throated martens (Martes flavigula). We chemically immobilized these species in Thailand using a mixture of ketamine hydrochloride with xylazine hydrochloride (KH–XH) and tiletamine hydrochloride with zolazepam hydrochloride (TH–ZH). Mean (±SD) dose of KH–XH was 18.1±5.3 and 0.9±0.1 mg/kg for dholes (n=2), 19.7±4.1 and 1.3±0.4 mg/kg for binturongs (n=8), and 28.7±3.3 and 1.1±0.1 mg/kg for yellow-throated martens (n=5), respectively. Mean dose of TH–ZH was 4.1±0.1 mg/kg for dhole (n=2). Induction time, duration of anesthesia, and recovery time were satisfactory for standard field research procedures including radio-collaring, although the effects on yellow-throated martens and binturongs were more variable. Respiration and muscle rigidity were monitored during sedation with no observed adverse physiological effects. Individuals were released after full recovery and monitored via radio telemetry for 4–23 months with no observed detrimental effects due to chemical immobilization. We conclude that KH–XH and TH–ZH are safe and effective immobilization agents for these carnivores; however, we suggest testing different KH–XH ratios and dosages, and other immobilizing agents for these species.     

Novel highly potent mu-opioid receptor antagonist based on endomorphin-2 structure

The mu-opioid agonists endomorphin-1 (Tyr-Pro-Trp-Phe-NH(2)) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) exhibit an extremely high selectivity for the mu-opioid receptor and thus represent a potential framework for modification into mu-antagonists. Here we report on the synthesis and biological evaluation of novel [d-2-Nal(4)]endomorphin-2 analogs, [Sar(2),d-2-Nal(4)]endomorphin-2 and [Dmt(1),Sar(2),d-2-Nal(4)]endomorphin-2 (Dmt=2'6'-dimethyltyrosine; Sar=N-methylglycine, sarcosine; d-2-Nal=3-(2-naphthyl)-d-alanine). [Dmt(1),Sar(2),d-2-Nal(4)]endomorphin-2 possessed very high affinity for the mu-opioid receptor (IC(50)=0.01+/-0.001 nM) and turned out to be a potent and extremely selective mu-opioid receptor antagonist, as judged by the in vitro aequorin luminescence-based calcium assay (pA(2)=9.19). However, in the in vivo hot plate test in mice this analog was less potent than our earlier mu-opioid receptor antagonist, [Dmt(1),d-2-Nal(4)]endomorphin-2 (antanal-2). The exceptional mu-opioid receptor in vitro activity and selectivity of [Dmt(1), Sar(2),d-2-Nal(4)]endomorphin-2 makes this analog a valuable pharmacological tool, but further modifications are needed to improve its in vivo profile.