PPARγ controls pregnancy outcome through activation of EG-VEGF: new insights into the mechanism of placental development.

PPARγ-deficient mice die at E9.5 due to placental abnormalities. The mechanism by which this occurs is unknown. We demonstrated that the new endocrine factor EG-VEGF controls the same processes as those described for PPARγ, suggesting potential regulation of EG-VEGF by PPARγ. EG-VEGF exerts its functions via prokineticin receptor 1 (PROKR1) and 2 (PROKR2). This study sought to investigate whether EG-VEGF mediates part of PPARγ effects on placental development. Three approaches were used: 1) in vitro, using human primary isolated cytotrophoblasts and the extravillous trophoblast cell line (HTR-8/SVneo); 2) ex vivo, using human placental explants (n = 46 placentas); and 3) in vivo, using gravid wild-type PPARγ(+/-) and PPARγ(-/-) mice. Major processes of placental development that are known to be controlled by PPARγ, such as trophoblast proliferation, migration, and invasion, were assessed in the absence or presence of PROKR1 and PROKR2 antagonists. In both human trophoblast cell and placental explants, we demonstrated that rosiglitazone, a PPARγ agonist, 1) increased EG-VEGF secretion, 2) increased EG-VEGF and its receptors mRNA and protein expression, 3) increased placental vascularization via PROKR1 and PROKR2, and 4) inhibited trophoblast migration and invasion via PROKR2. In the PPARγ(-/-) mouse placentas, EG-VEGF levels were significantly decreased, supporting an in vivo control of EG-VEGF/PROKRs system during pregnancy. The present data reveal EG-VEGF as a new mediator of PPARγ effects during pregnancy and bring new insights into the fine mechanism of trophoblast invasion.

Inhibitory effect of positively charged triazine antagonists of prokineticin receptors on the transient receptor vanilloid type-1 (TRPV1) channel.

Four positively charged compounds, previously shown to produce analgesic activity by interacting with prokineticin receptor or T-type calcium channels, were tested for their ability to inhibit capsaicin-induced elevation of intracellular Ca(2+) in HEK-293 cells stably transfected with the human recombinant TRPV1, with the goal of identifying novel TRPV1 open-pore inhibitors. KYS-05090 showed the highest potency as a TRPV1 antagonist, even higher than that of the open-pore triazine inhibitor 8aA. The latter showed quite remarkable agonist/desensitizer activity at the rat recombinant TRPM8 channel. The activity of KYS-05090 and the other compounds was selective because none of these compounds was able to modulate the rat TRPA1 channel. Open-pore inhibitors of TRPV1 may be a new class of multi-target analgesics with lesser side effects, such as loss of acute pain sensitivity and hyperthermia, than most TRPV1 antagonists developed so far.

Synthesis of sulfonamides incorporating piperazinyl-ureido moieties and their carbonic anhydrase I, II, IX and XII inhibitory activity.

By using SLC-0111 (4-fluorophenylureido-benzenesulfonamide), a sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor in Phase I clinical trials as an antitumor agent as lead molecule, a series of benzenesulfonamide derivatives incorporating ureido moieties was synthesized. The new compounds contain a 4-N-substituted piperazine fragment in which the ureido linker has been included, and were tested as inhibitors of the cytosolic human (h) hCA I and II isoforms, as well as the transmembrane, tumor-associated enzymes hCA IX and XII. Depending on the substitution pattern at the piperazine ring, low nanomolar inhibitors were detected against all four isoforms, making the new class of sulfonamides of interest for various pharmacologic applications.

Synthesis and carbonic anhydrase I, II, IX and XII inhibitory activity of sulfamates incorporating piperazinyl-ureido moieties.

A series of sulfamates were synthesized using as lead compound SLC-0111, a sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor in Phase I clinical trials. The new derivatives incorporated ureido moieties as spacers between the benzene sulfamate fragment which binds the zinc ion from the active site, and the tail of the inhibitor, but the urea moieties were part of a substituted piperazine ring system. The derivatives (and some of their phenol precursors) were tested for the inhibition of the cytosolic, hCA I and II (off target isoforms) and the trans-membrane, tumor-associated hCA IX and XII enzymes (anticancer drug targets). Generally hCA I was not effectively inhibited, whereas many low nanomolar inhibitors were evidenced against hCA II (KIs in the range of 1.0-94.4 nM), IX (KIs in the range of 0.91-36.9 nM), and XII (KIs in the range of 1.0-84.5 nM). The best substitution fragments at the piperazine ring included the following moieties: 3-methylphenyl, 2,3-dimethylphenyl, 4-methoxyphenyl, 6-arylpyrimidine-2-yl.

Synthesis and carbonic anhydrase I, II, IX and XII inhibition studies of 4-N,N-disubstituted sulfanilamides incorporating 4,4,4-trifluoro-3-oxo-but-1-enyl, phenacylthiourea and imidazol-2(3H)-one/thione moieties.

A series of sulfonamides incorporating the sulfanilamide (SA) scaffold were prepared. Reaction of the 4-amino moiety of SA with benzyl chlorides or substituted bromoacetophenones afforded the 4-mono-alkylated derivatives which were then reacted with 1,1,1-trifluoro-4-isobutoxybut-3-en-2-one leading to a series of 4-N,N-disubstituted SAs. The key intermediates were also reacted with ethoxycarbonyl isothiocyanate leading to thioureas or were cyclized in the presence of potassium cyanate/isothiocyanate to the corresponding imidazol-2(3H)-one/thiones. The new compounds were tested as inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, and the transmembrane, tumor-associated CA IX and XII. These sulfonamides were ineffective CA I and II inhibitors but were nanomolar CA IX and XII inhibitors, making them of interest as clinical candidates for antitumor/antimetastasis applications.

Synthesis and biological evaluation of novel acylhydrazone derivatives as potential antitumor agents.

We have designed, synthesized, and evaluated as potential antitumor agents a series of 2-hydroxybenzylidene derivatives of the N-(2-trifluoromethylpiridyn-4-yl)anthranilic acid hydrazide, and some analogues bearing a (2-trifluoromethyl)piridyn-4-ylamino group in 3- or 4-position of benzohydrazide or 4-position of phenylacetohydrazide. Compounds 12e, 13e, 15e, and 16e, bearing a 4-(diethylamino)salicylidene group exhibited potent cytotoxicity, with averaged GI50 values in sub-micromolar range, and a variety of cell selectivity at nanomolar concentrations. The determination of acute toxicity in athymic nudes mice proved some compounds to be non-toxic, making them good candidates for further study as antitumor agents.

Inhibitory properties of ibuprofen and its amide analogues towards the hydrolysis and cyclooxygenation of the endocannabinoid anandamide.

A dual-action cyclooxygenase (COX)-fatty acid amide hydrolase (FAAH) inhibitor may have therapeutic usefulness as an analgesic, but a key issue is finding the right balance of inhibitory effects. This can be done by the design of compounds exhibiting different FAAH/COX-inhibitory potencies. In the present study, eight ibuprofen analogues were investigated. Ibuprofen (1), 2-(4-Isobutylphenyl)-N-(2-(3-methylpyridin-2-ylamino)-2-oxoethyl)propanamide (9) and N-(3-methylpyridin-2-yl)-2-(4'-isobutylphenyl)propionamide (2) inhibited FAAH with IC(50) values of 134, 3.6 and 0.52 µM respectively. The corresponding values for COX-1 were ~29, ~50 and ~60 µM, respectively. Using arachidonic acid as substrate, the compounds were weak inhibitors of COX-2. However, when anandamide was used as COX-2 substrate, potency increased, with approximate IC(50) values of ~6, ~10 and ~19 µM, respectively. Compound 2 was confirmed to be active in vivo in a murine model of visceral nociception, but the effects of the compound were not blocked by CB receptor antagonists.

Flavones and structurally related 4-chromenones inhibit carbonic anhydrases by a different mechanism of action compared to coumarins.

An inhibition study of several carbonic anhydrase (CA, EC 4.2.1.1) isoforms with flavones and aminoflavones, compounds possessing a rather similar scaffold with the coumarins, recently discovered inhibitors of this enzyme, is reported. The natural product flavone and some of its hydroxylated derivatives did not show time-dependent inhibition of the CAs, sign that they are not hydrolyzed within the enzyme active site as the (thio)coumarins and lactones. These compounds were low micromolar inhibitors of hCA I, II, IX and XII, with K(I)s in the range of 1.88-9.07 µM. A series of substituted 2-amino-3-phenyl-4H-chromen-4-ones, incorporating chloro- and methoxy substituents in various positions of the heterocycle, were then prepared and assayed as hCA I and II inhibitors, showing activity in the micromolar range. Some of these derivatives, as well as cis+trans resveratrol, were then assayed for the inhibition of all catalytically active mammalian CA isoforms, hCA I, II, III, IV, VA, VB, VI, VII, IX, XII, XIII, XIV and mCA XV (h=human, m=murine enzyme). These derivatives inhibited these CAs in the submicromolar-low micromolar range. Flavones, although not as active as the coumarins, may be considered as interesting leads for the design of non-sulfonamide CA inhibitors.

Synthesis and in vitro antitumor activity of new 4,5-dihydropyrazole derivatives.

A series of 3,5-diaryl-4,5-dihydropyrazole regioisomers, and their 1-acetylated derivatives, bearing a 3,4,5-trimethoxyphenyl moiety combined with a variety of substituted phenyl rings, was synthesized and evaluated for antitumor activity. Results of the in vitro assay against a non-small cell lung carcinoma cell line (NCI-H460) showed several compounds to be endowed with cytotoxicity in micromolar to sub-micromolar range, depending on substitution pattern and position of aryl rings on 4,5-dihydropyrazole core. Potent and selective activity was also observed in the NCI 60 human cancer cell line panel. 5-(3,4,5-Trimethoxyphenyl)pyrazolines 31 and 39 were found to possess potent antiproliferative activity against SR and MDA-MB-435, with GI(50) inhibitory values in nanomolar range. Structure-activity relationships revealed that introduction of a (hydroxy)acetyl group at N-1 of inactive 5-(3,4,5-trimethoxyphenyl)pyrazolines, results in a clear in vitro activating effect. Compound 31 (IC(50)=5.16 microM) showed inhibition of tubulin polymerization comparable to that of CA-4 (IC(50)=4.92 microM).

Synthesis and evaluation of anticancer activity of 2-arylamino-6-trifluoromethyl-3-(hydrazonocarbonyl)pyridines.

The synthesis and anticancer activity of 2-arylamino-6-trifluoromethyl-3-(hydrazonocarbonyl)pyridines is described. The new trifluoromethylpyridine derivatives were evaluated for their anticancer activity toward human cancer cell lines by the National Cancer Institute (NCI). Most of them had excellent growth inhibition activity, having GI(50) values in the low micromolar to nanomolar concentration range. The most potent 2,6-dichlorobenzaldehydehydrazone 29 inhibited the growth of all tested cancer cell lines with nanomolar potency, and did not show animal toxicity. Hydrazone 29 has been selected by the Biological Evaluation Committee of NCI for testing in vivo Hollow Fiber Assay.

Design, synthesis, and in vitro antitumor activity of new 1,4-diarylimidazole-2-ones and their 2-thione analogues.

A series of new 1,4-diarylimidazol-2(3H)-one derivatives and their 2-thione analogues has been prepared and evaluated in vitro for antitumor activity against the NCI human cancer cell panel. Compounds bearing a 3,4,5-trimethoxyphenyl ring linked to either N-1 or C-4 position of the imidazole core demonstrated an interesting profile of cytotoxicity with preferential activity against leukemic cell lines. Compound 13 exhibited a potent antitumor activity against MOLT-4 (GI(50)=20 nM) and SR (GI(50)=32 nM) cell lines.

Synthesis and evaluation of antitumoral activity of ester and amide derivatives of 2-arylamino-6-trifluoromethyl-3-pyridinecarboxylic acids.

The synthesis and antitumoral activity of ester and amide derivatives of 2-arylamino-6-trifluoromethyl-3-pyridinecarboxylic acids 8-58 is described. Trifluoromethylpyridine derivatives 8-58 were evaluated for their anticancer activity toward human tumoral cell lines by the National Cancer Institute (NCI). Most of them possess encouraging anticancer activity, having GI(50) values in the low micromolar to nanomolar concentration range. The 3,4,5-trimethoxyphenylamide 44 was the most active, and it is now under review by NCI Biological Evaluation Committee for possible further studies.

Effect of lysine at C-terminus of the Dmt-Tic opioid pharmacophore.

Substitution of Gly with side-chain-protected or unprotected Lys in lead compounds containing the opioid pharmacophore Dmt-Tic [H-Dmt-Tic-Gly-NH-CH(2)-Ph, mu agonist/delta antagonist; H-Dmt-Tic-Gly-NH-Ph, mu agonist/delta agonist; and H-Dmt-Tic-NH-CH(2)-Bid, delta agonist (Bid = 1H-benzimidazole-2-yl)] yielded a new series of compounds endowed with distinct pharmacological activities. Compounds (1-10) included high delta- (Ki(delta) = 0.068-0.64 nM) and mu-opioid affinities (Ki(mu) = 0.13-5.50 nM), with a bioactivity that ranged from mu-opioid agonism {10, H-Dmt-Tic-NH-CH[(CH2)4-NH2]-Bid (IC50 GPI = 39.7 nM)} to a selective mu-opioid antagonist [3, H-Dmt-Tic-Lys-NH-CH2-Ph (pA2(mu) = 7.96)] and a selective delta-opioid antagonist [5, H-Dmt-Tic-Lys(Ac)-NH-Ph (pA2(delta) = 12.0)]. The presence of a Lys linker provides new lead compounds in the formation of opioid peptidomimetics containing the Dmt-Tic pharmacophore with distinct agonist and/or antagonist properties.

New potential anticancer agents based on the anthranilic acid scaffold: synthesis and evaluation of biological activity.

The synthesis and anticancer activity of new compounds designed on the anthranilic acid scaffold are reported. The antiproliferative activity was assayed by the National Cancer Institute in established in vitro and in vivo anticancer experimental models. Structural variations based on the flufenamic acid motif afforded a series of (hetero)aryl esters of N-(2-(trifluoromethyl)pyridin-4-yl)anthranilic acid, which showed in vitro growth inhibitory properties against human tumor cell lines in nanomolar to low micromolar concentrations. The pyridinyl ester 25 exhibited very potent in vitro antiproliferative efficacy, with a chemosensitive profile showing a number of GI(50) values at concentrations lower than 10(-7) M in the full panel of human tumor cell lines. Compound 25 was also tested in vivo as a potential anticancer agent in the hollow fiber assay and in human tumor xenografts, showing moderate inhibitory properties. Analysis of biological activities and the COMPARE procedure was utilized to support putative biochemical mechanisms implicated with the antiproliferative activity.

In vitro antimycobacterial activity of newly synthesised S-alkylisothiosemicarbazone derivatives and synergistic interactions in combination with rifamycins against Mycobacterium avium.

The antimycobacterial activities of two new S-alkylisothiosemicarbazone derivatives (1i and 1f) against 32 Mycobacterium avium isolates were investigated. The minimum inhibitory concentrations (MICs) were significantly lower than those of rifampicin and other reference drugs. The two derivatives also showed excellent intracellular activity against M. avium residing in the macrophage-like J774 cells. Interestingly, the combination of subinhibitory concentrations of 1i and rifabutin or rifampicin induced a potent synergistic effect, as determined by the fractional inhibitory concentration indexes (FICIs) ranging between 0.103 and 0.412. Such synergistic effect resulted in a 81-fold and 139-fold reduction of the MICs of rifabutin and rifampicin, respectively. Enhancement of intracellular activity of rifabutin by the S-alkylisothiosemicarbazone derivative 1i was also observed. Results indicate that S-alkylisothiosemicarbazones can be useful in the therapy and prophylaxis of M. avium infections and can represent a template for the development of novel antimycobacterial drugs. Furthermore, as a consequence of their ability to enhance the activity of rifamycins, a reduction of drug interactions following the co-administration of protease inhibitors could be achieved by lower doses of rifampicin and rifabutin.

Synthesis and antiproliferative activity of 2,6-dibenzylamino-3,5-dicyanopyridines on human cancer cell lines.

A new series of 2,6-dibenzylamino-3,5-dicyanopyridines were synthesized and evaluated for their in vitro anticancer activity toward cell lines of nine different types of human cancer. Some of newly prepared compounds demonstrated remarkable anticancer activity against most of the tested subpanel tumor cell lines.

TRPV1 modulators: Synthesis and in vitro evaluation of 1-heteroaryl piperidinecarboxamide and piperazinylurea derivatives.

A series of new 1-heteroaryl piperidinecarboxamide and piperazinylurea derivatives was synthesized and evaluated as TRPV1 modulators in a Ca(2+) channel assay in HEK-293 cells overexpressing the human recombinant TRPV1 channel. Structural variations in the putative key portions of the molecules afforded several compounds endowed with agonist and/or antagonist/desensitizing activity at low micromolar concentration. As promising examples from this series, the piperidine-3-carboxamide derivative 31 exerts agonist/desensitizing activity at low micromolar concentration, while piperazinylurea derivatives 39 and 41 act as antagonists with sub-micromolar potency.

Design, synthesis, and anti-HIV-1 activity of 1-substituted 3-(3,5-dimethylbenzyl)triazine derivatives.

BACKGROUND: The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is an attractive target for the development of drugs used in the treatment of HIV-1 infection and acquired immune deficiency syndrome (AIDS). We have continued the search for novel anti-HIV-1 agents using the structure-activity relationships of the successful 1,3-disubstituted and 1,3,6-trisubstituted uracil-type HIV-1 RT inhibitors. METHODS: A series of new triazine analogs were synthesized using an established method. The anti-HIV-1 activities of these compounds were determined based on the inhibition of virus-induced cytopathogenicity in MT-4 cells. The cytotoxicity of the compounds was evaluated by assessing the viability of mock-infected cells. RESULTS: Some of the compounds showed good-to-moderate activities against HIV-1, with half-maximal effective concentrations (EC50) in the submicromolar range. In particular, a dihydro-1-(4-aminobenzyl)triazine analog showed satisfactory anti-HIV-1 activity with an EC50 of 0.110 µM and a selectivity index (SI) of 909. Furthermore, molecular modeling analyses were performed to explore the major interactions between HIV-1 RT and potent inhibitors. These results may be important for further development of this class of compounds as anti-HIV-1 agents. CONCLUSION: The satisfactory anti-HIV-1 activity of triazine analogs may serve as the basis for further investigations of the behavior of this class of compounds against drug-resistant mutants.

Characterisation of (R)-2-(2-Fluorobiphenyl-4-yl)-N-(3-Methylpyridin-2-yl)Propanamide as a Dual Fatty Acid Amide Hydrolase: Cyclooxygenase Inhibitor.

BACKGROUND: Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-2) inhibitors is a promising approach for pain-relief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl)-N-(3-methylpyridin-2-yl)propanamide (Flu-AM1). These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-2 for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-2-derived lipids in intact cells are not known. METHODOLOGY/PRINCIPAL FINDINGS: COX inhibition was determined using an oxygraphic method with arachidonic acid and 2-arachidonoylglycerol (2-AG) as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA) as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Both enantiomers inhibited COX-2 in a substrate-selective and time-dependent manner, with IC50 values in the absence of a preincubation phase of: (R)-Flu-AM1, COX-1 (arachidonic acid) 6 µM; COX-2 (arachidonic acid) 20 µM; COX-2 (2-AG) 1 µM; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 µM; COX-2 (arachidonic acid) 10 µM; COX-2 (2-AG) 0.7 µM. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 µM) greatly inhibited the production of prostaglandin D2 and E2 in both unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R)-Flu-AM1 or by 10 µM flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 µM). CONCLUSIONS/SIGNIFICANCE: Both enantiomers of Flu-AM1 are more potent inhibitors of 2-AG compared to arachidonic acid oxygenation by COX-2. Inhibition of COX in lipopolysaccharide + interferon γ- stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the large induction of COX-2 produced by this treatment.

Interaction of the N-(3-Methylpyridin-2-yl)amide Derivatives of Flurbiprofen and Ibuprofen with FAAH: Enantiomeric Selectivity and Binding Mode.

BACKGROUND: Combined fatty acid amide hydrolase (FAAH) and cyclooxygenase (COX) inhibition is a promising approach for pain-relief. The Flu-AM1 and Ibu-AM5 derivatives of flurbiprofen and ibuprofen retain similar COX-inhibitory properties and are more potent inhibitors of FAAH than the parent compounds. However, little is known as to the nature of their interaction with FAAH, or to the importance of their chirality. This has been explored here. METHODOLOGY/PRINCIPAL FINDINGS: FAAH inhibitory activity was measured in rat brain homogenates and in lysates expressing either wild-type or FAAH(T488A)-mutated enzyme. Molecular modelling was undertaken using both docking and molecular dynamics. The (R)- and (S)-enantiomers of Flu-AM1 inhibited rat FAAH with similar potencies (IC50 values of 0.74 and 0.99 µM, respectively), whereas the (S)-enantiomer of Ibu-AM5 (IC50 0.59 µM) was more potent than the (R)-enantiomer (IC50 5.7 µM). Multiple inhibition experiments indicated that both (R)-Flu-AM1 and (S)-Ibu-AM5 inhibited FAAH in a manner mutually exclusive to carprofen. Computational studies indicated that the binding site for the Flu-AM1 and Ibu-AM5 enantiomers was located between the acyl chain binding channel and the membrane access channel, in a site overlapping the carprofen binding site, and showed a binding mode in line with that proposed for carprofen and other non-covalent ligands. The potency of (R)-Flu-AM1 was lower towards lysates expressing FAAH mutated at the proposed carprofen binding area than in lysates expressing wild-type FAAH. CONCLUSIONS/SIGNIFICANCE: The study provides kinetic and structural evidence that the enantiomers of Flu-AM1 and Ibu-AM5 bind in the substrate channel of FAAH. This information will be useful in aiding the design of novel dual-action FAAH: COX inhibitors.