Breastmilk PUFA strongly associated with maternal dietary intake but not anthropometric parameters and breastmilk carotenoids.

BACKGROUND: Long chain polyunsaturated fatty acids (PUFA) and the optimal n-6/n-3 fatty acids ratio are essential for proper neurodevelopment in infancy. This study aimed to evaluate the association between breastmilk fatty acid intake and maternal dietary intake, anthropometrics and breastmilk carotenoid levels. METHODS: This observational, prospective study included 44 women in the first, third, and sixth month of lactation. At each study visit, maternal anthropometric measures were assessed and breastmilk samples were collected and assessed for fatty acids and carotenoids. At the third and sixth month, maternal diet was evaluated by three-day foods record. RESULTS: Mean breastmilk docosahexaenoic (DHA) was 0.58%, 0.47%, and 0.49%, respectively at the 1, 3, and 6 month (p ≤ 0.05). Mean DHA intake were higher in month 3 compared to 6: 357 vs. 169 mg/day. Pre-pregnancy BMI was associated with SFA, PUFA, and n-6 PUFA at 1 month, whereas current BMI to SFA at months 1 and 3. DHA was correlated with lycopene, total carotenoids at 1 month and total carotenoids at month 3, whereas n-3 PUFA to lycopene at 1 month. DHA, n-3 PUFA, n-6 PUFA and saturated (SFA) levels were associated with its dietary intake both at months 3 and 6, AA/DHA and LA/ALA ratios only at month 3. CONCLUSIONS: Maternal intake of PUFA and n-6/n-3 ratios were a good predictor of its breastmilk composition, whereas pre-pregnancy and current BMI, as well as breastmilk carotenoids had a limited influence.

Gut response to pasteurized donor human milk in a porcine model of the premature infant.

This study investigated the tolerance and safety of pasteurized donor human milk (PDHM) given either alone or together with commercially-used supplements in a porcine model of premature infants. A porcine model, mimicking human neonates at 30-32 weeks of gestational age, was used. The 7-day experiment was performed on 20 piglets. After birth, the piglets were infused with porcine immunoglobulins via the umbilical artery and surgically fitted with a stomach port. The piglets were then randomized into five groups and fed either PDHM, different variants of fortified PDHM or 'raw' human milk (RHM). Preterm piglets fed PDHM showed signs of gastrointestinal intolerance. Four piglets across the various PDHM-fed groups died, none of them were from the group fed PDHM supplemented with long-chain polyunsaturated fatty acids (LC PUFA). In all groups fed PDHM, macroscopic features of enterocolitis were observed, however, these pathological gut changes were less manifested in piglets receiving PDHM supplemented with LC PUFA. The piglets fed RHM had no specific signs of gut damage. The poor tolerance to PDHM suggests changes in milk composition caused by the Holder pasteurization. The supplementation with LC PUFA probably improves tolerance to PDHM.

Circulating CD3+56+ cell subset in pre-diabetes.

The CD3+56+ cells are a small but significant population of T lymphocytes encompassing NKT-like and NKT cells, which may play the essential role at the very early stages of atherosclerotic plaque development. The frequency and activity of CD3+56+ cells in atherosclerosis-inducing dysglycaemic disease (diabetes type 2 or pre-diabetes) is largely unknown.We analysed CD3+56+ cell count, granzyme, perforin and annexin V profiles in the peripheral blood from a group of patients with pre-diabetes, with diabetes type 2 and from non-dysglycaemic controls. Measurements were made of fasting glucose levels, HbA1c, 1,5-anhydroglucitol and lipid profile.The mean counts of CD3+56+ cells were significantly higher in patients with pre-diabetes compared to both patients with diabetes and to control group. There was an increase in the number of CD3+56+ cells producing granzyme and perforin in pre-diabetic patients compared to other groups, while there were no difference in annexin V+ populations within examined groups. It was confirmed that CD3+56+ cells count is modified by metabolic factors and their parameters, namely HbA1c and 1,5-anhydroglucitol values.It could be stated that the alterations of CD3+ 56+ cells count in peripheral blood of pre-diabetic and type 2 diabetic patients are related to different grades of carbohydrate deteriorations - postprandial hyperglycaemia and chronic hyperglycaemia.

ADN-1184 a monoaminergic ligand with 5-HT(6/7) receptor antagonist activity: pharmacological profile and potential therapeutic utility.

BACKGROUND AND PURPOSE: Many dementia patients exhibit behavioural and psychological symptoms (BPSD) that include psychosis, aggressivity, depression and anxiety. Antipsychotic drugs are frequently prescribed but fail to significantly attenuate mood deficits, may interfere with cognitive function and are associated with motor and cardiac side effects, which are problematic in elderly patients. A need therefore exists for drugs that are better suited for the treatment of BPSD. EXPERIMENTAL APPROACH: We used in vitro cellular and in vivo behavioural tests to characterize ADN-1184, a novel arylsulfonamide ligand with potential utility for treatment of BPSD. KEY RESULTS: ADN-1184 exhibits substantial 5-HT6 /5-HT7 /5-HT2A /D2 receptor affinity and antagonist properties in vitro. In tests of antipsychotic-like activity, it reversed MK-801-induced hyperactivity and stereotypies and inhibited conditioned avoidance response (MED = 3 mg·kg(-1) i.p.). Remarkably, ADN-1184 also reduced immobility time in the forced swim test at low doses (0.3 and 1 mg·kg(-1) i.p.; higher doses were not significantly active). Notably, up to 30 mg·kg(-1) ADN-1184 did not impair memory performance in the passive avoidance test or elicit significant catalepsy and only modestly inhibited spontaneous locomotor activity (MED = 30 mg·kg(-1) i.p.). CONCLUSIONS AND IMPLICATIONS: ADN-1184 combines antipsychotic-like with antidepressant-like properties without interfering with memory function or locomotion. This profile is better than that of commonly used atypical antipsychotics tested under the same conditions and suggests that it is feasible to identify drugs that improve BPSD, without exacerbating cognitive deficit or movement impairment, which are of particular concern in patients with dementia.

Characterization and differential expression of cathepsin L3 alleles from Fasciola hepatica.

Fasciola hepatica infections cause significant global problems in veterinary and human medicine, including causing huge losses in cattle and sheep production. F. hepatica host infection is a multistage process and flukes express papain-like cysteine proteases, termed cathepsins, which play pivotal roles in virulence through host entry, tissue migration and immune evasion. Expression of these proteases is developmentally regulated. Recent studies indicate that excystment of infective larvae is dependent on cysteine proteases and together FhCL3 and FhCB account for over 80% of total protease activity detectable in newly excysted juvenile (NEJ) fluke. This paper focuses on members of the cathepsin L gene family, specifically those belonging to the CL3 clade. The cDNA of two novel cathepsin L3 proteases--FhCL3-1 and FhCL3-2 were cloned. The mRNA transcript expression levels for these enzymes were significantly different at various time points in life development stages obtained in vitro, from dormant metacercariae to NEJ 24h after excystment. Maximum expression levels were observed in NEJ immediately after excystment. In all stages examined by Real Time PCR, FhCL3-2 was expressed at a higher level compared to FhCL3-1 which was expressed only at very low levels. Western blot and immunohistochemical analysis also indicated higher expression of the FhCL3-2 allele and its secretory nature. The ability of antibody responses from rats and sheep challenged with F. hepatica to recognize recombinant FhCL3-1 and FhCL3-2 was shown to differ. Differences were also confirmed through the use of anti-rFhCL3-1 and anti-rFhCL3-2 sera in Western blot analysis of juvenile excretory/secretory (ES) material separated by 2D electrophoresis. These results indicate analysis of relative expression of parasite virulence factors from different populations is required, as this will likely impact the effectiveness of vaccines based on these antigens.

Cost-effective multiplexing before capture allows screening of 25 000 clinically relevant SNPs in childhood acute lymphoblastic leukemia.

Genetic variants, including single-nucleotide polymorphisms (SNPs), are key determiners of interindividual differences in treatment efficacy and toxicity in childhood acute lymphoblastic leukemia (ALL). Although up to 13 chemotherapeutic agents are used in the treatment of this cancer, it remains a model disease for exploring the impact of genetic variation due to well-characterized cytogenetics, drug response pathways and precise monitoring of minimal residual disease. Here, we have selected clinically relevant genes and SNPs through literature screening, and on the basis of associations with key pathways, protein-protein interactions or downstream partners that have a role in drug disposition and treatment efficacy in childhood ALL. This allows exploration of pathways, where one of several genetic variants may lead to similar clinical phenotypes through related molecular mechanisms. We have designed a cost-effective, high-throughput capture assay of ∼25,000 clinically relevant SNPs, and demonstrated that multiple samples can be tagged and pooled before genome capture in targeted enrichment with a sufficient sequencing depth for genotyping. This multiplexed, targeted sequencing method allows exploration of the impact of pharmacogenetics on efficacy and toxicity in childhood ALL treatment, which will be of importance for personalized chemotherapy.

Microglia-derived TGF-beta as an important regulator of glioblastoma invasion--an inhibition of TGF-beta-dependent effects by shRNA against human TGF-beta type II receptor.

The invasion of tumor cells into brain tissue is a pathologic hallmark of malignant gliomas and contributes to treatment failures. Diffuse glioblastomas contain numerous microglial cells, which enhance the progression of gliomas; however, factors responsible for invasion-promoting role of microglia are unknown. Transforming growth factor-beta (TGF-beta) can enhance tumor growth, invasion, angiogenesis and immunosuppression. Antagonizing TGF-beta activity has been shown to inhibit tumor invasion in vitro and tumorigenicity, but a systemic inhibition or lack of TGF-beta signaling results in acute inflammation and disruption of immune system homeostasis. We developed plasmid-transcribed small hairpin RNAs (shRNAs) to downregulate the TGF-beta type II receptor (TbetaIIR) expression, which effectively inhibited cytokine-induced signaling pathways and transcriptional responses in transiently transfected human glioblastoma cells. Silencing of TbetaIIR abolished TGF-beta-induced glioblastoma invasiveness and migratory responses in vitro. Moreover, tumorigenicity of glioblastoma cells stably expressing TbetaIIR shRNAs in nude mice was reduced by 50%. Microglia strongly enhanced glioma invasiveness in the co-culture system, but this invasion-promoting activity was lost in glioma cells stably expressing shTbetaRII, indicating a crucial role of microglia-derived TGF-beta in tumor-host interactions. Our results demonstrate a successful targeting of TGF-beta-dependent invasiveness and tumorigenicity of glioblastoma cells by RNAi-mediated gene silencing.

Analgesic and sedative activities of lactucin and some lactucin-like guaianolides in mice.

Lactucin (1) and its derivatives lactucopicrin (2) and 11beta,13-dihydrolactucin (3), which are characteristic bitter sesquiterpene lactones of Lactuca virosa and Cichorium intybus, were evaluated for analgesic and sedative properties in mice. The compounds showed analgesic effects at doses of 15 and 30 mg/kg in the hot plate test similar to that of ibuprofen, used as a standard drug, at a dose of 30 mg/kg. The analgesic activities of the compounds at a dose of 30 mg/kg in the tail-flick test were comparable to that of ibuprofen given at a dose of 60 mg/kg. Lactucopicrin appeared to be the most potent analgetic of the three tested compounds. Lactucin and lactucopicrin, but not 11beta,13-dihydrolactucin, also showed sedative properties in the spontaneous locomotor activity test.

Polyoma BK virus reactivation in kidney and pancreas-kidney recipients.

BK virus infection has become important factor affecting graft function in renal transplant recipients. One of the most important complication of BK infection is nephropathy in patients after renal transplantation. The aim of this study was to evaluate incidence of BK reactivation and nephropathy in our population of renal allograft recipients. One hundred twelve renal or pancreas-kidney allograft recipients were included for the 24 months follow-up. The incidence of BK nephropathy was 7.85% and viremia 27.96%. In the second study group there were 28 patients with graft function deterioration evaluated at the time of biopsy. In this group incidence of BK nephropathy was 7.1% and viral reactivation was diagnosed in 10.7% of patients. In our center, the incidence of BK nephropathy is the same as worldwide. The risk of BK virus replication is highest during first 15 months after the surgical procedure.

Transplantation of hepatocytes: temporary elimination of scavenger cells prevents early loss of grafted cells.

Transplants of isolated syngeneic and allogeneic hepatocytes are rapidly disintegrated, irrespective of the site of engraftment; be it spleen, liver, portal vein, peritoneum, or subcutaneous tissue. Host scavenger cells are responsible for this reaction. We designed a method overcoming early disintegration of the grafted hepatocytes. It consisted of administration of anti-asialoGM1 antiserum eliminating natural killer cells; sublethal whole body irradiation; and reconstitution with syngeneic bone marrow cells, ligation of host bile duct, intrasplenic hepatocyte transplantation, and three consecutive partial hepatectomies. Six months after transplantation a glycogen-rich, trabeculae-forming, dividing hepatocytes, situated along strands of newly-formed fibrous tissue and numerous dilated blind bile cannaliculae were observed. There was evidently more bile canaliculae in hosts with ligated bile duct than nonligated controls. This is the first study showing fibrous tissue formed at the site of hepatocyte implantation, and stellate cells are presumably involved in this process.

Polyomavirus BK infection.

Because it is an important factor affecting renal transplant function, BK infections are significant problem in posttransplant. BK nephropathy develops in 5% of renal allograft recipients, in most cases within the first year after the procedure. The gold standard for BK nephropathy diagnosis is still immunohistochemical staining for large T antigen in graft biopsy specimens. The aim of the present study was to evaluate the incidence of and factors influencing BK nephropathy in our renal allograft population. Among 89 renal or pancreas/kidney allograft recipients, BKV DNA was detected in 1 or more serum samples in 17 patients but BK nephropathy was diagnosed in only 1 case. Plasmacytic tubulitis was an exclusive feature in PCR-positive patients with 2 (20%) cases but no such findings in the PCR-negative group. In 40% of patients in the PCR-positive group at least 1 rejection episode was diagnosed versus 22% in the PCR-negative group. There were no significant differences in both groups according to total ischemia time, immunosuppressive treatments, or mean serum creatinine at 1 year after transplantation.

Transplantation of hepatocytes: elimination of recipient natural killer cells with irradiation and bone marrow reconstitution prevent early graft dysfunction.

Transplanted isolated syngeneic and allogeneic hepatocytes rapidly disintegrate, irrespective of the origin or the site of engraftment namely spleen, liver, portal vein, peritoneum, or subcutaneous tissues. Although various methods have been applied to attenuate this reaction, none have been found effective. We applied a combined protocol consisting of administration of anti-asialoGM1 antiserum (eliminating NK cells), sublethal whole-body irradiation, and reconstitution with syngeneic bone marrow cells to intrasplenic hepatocyte transplantation and 3 consecutive partial hepatectomies. This method overcame the early disintegration of grafted hepatocytes. Ninety days after transplantation numerous hepatocyte clusters and dilated bile canaliculae, occupying two thirds of the spleen, were observed, with some hepatocytes adhering to the bile ducts forming Hering's canals. Mitotic figures were noticed. There were no recipient mononuclear infiltrates around the hepatocyte clusters.

2-H- and 2-acyl-9- [omega-[4-(2-methoxyphenyl)piperazinyl]-alkyl]-1,2,3,4-tetrahydro-beta-carbolines as ligands of 5-HT1A and 5-HT2A receptors.

Three series of new unsubstituted or 2-acyl 1,2,3,4-tetrahydro-beta-carbolines (THBC), connected to 1-(o-methoxyphenyl)piperazine by 2-, 3- or 4-membered alkylene spacer (3, 4 or 5, respectively) in position 9, were synthesized and their 5-HT1A/5-HT2A receptor affinities and functional in vivo activities were investigated. Radioligand binding studies showed that unsubstituted (a) and acyl (b-f) derivatives with prop-1,3-ylene (4) and particularly with but-1,4-ylene (5) spacer had a high 5-HT1A receptor affinity (Ki = 30-110 nM), whereas the 5-HT1A affinity of derivatives with ethylene spacer (3) was low. All those compounds (except 5c, Ki = 44 nM) did not distinctly bind to 5-HT2A receptors. The obtained results indicated that the length of an alkylene chain was a crucial parameter for determining 5-HT1A receptor affinities of the tested compounds, while acyl substituents in position 2 of THBC were not important for their 5-HTIA/5-HT2A activities. It was also demonstrated that the few selected compounds (4d, 5a-c and 5e) with the highest affinity (Ki up to 50 nM) for 5-HT1A receptors, administered at doses of 10-20 mg/kg, behaved like antagonists of postsynaptic 5-HT1A receptors, as they reduced the 8-OH-DPAT (5-HT1A agonist)-induced lower lip retraction and behavioral syndrome in rats. Moreover, 4d seemed to be an agonist of presynaptic 5-HT1A receptors, since the hypothermia induced by its administration was attenuated by WAY 100635 (5-HT1A antagonist). Compound 5c, 5-HT2A receptor ligand, demonstrated an antagonistic activity, as it inhibited the (+/-)DOI (5-HT2A agonist)-induced head twitches in mice. The obtained results of in vivo studies suggest that introduction of different acyl substituents in position 2 of THBC with propylene or butylene spacer between tricyclous and arylpiperazine moiety is insignificant for the postsynaptic 5-HTIA receptor activity of the compounds tested in vivo. On the other hand, only compound 5c with an acryloyl group and a butylene chain behaved like a 5-HT1A/5-HT2A antagonist.

Substitution mode of the amide fragment in some new N-[omega-[4-(2-methoxyphenyl)piperazin-1-yl]alkyl]pyrid-2(1H)-ones and their 5-HT1A/5-HT2A activity.

A series of omega-[4-(2-methoxyphenyl)piperazin-1-yl]alkyl derivatives with terminal pyrid-2(1H)-one fragments was synthesized and evaluated for their 5-HTIA and 5-HT2A activity. Enlargement of the aromatic amide system by its substitution with phenyl and/or p-methoxyphenyl in positions 4, 5 and/or 6, as well as modification of an aliphatic spacer allowed us to better understand structure-activity relationships in that group of compounds. The results of in vitro and in vivo experiments showed that only unsubstituted (1b) and monosubstituted (2b-4b) derivatives with the tetramethylene spacer demonstrated high 5-HTIA receptor affinity (Ki = 15-40 nM) and 5-HT1A/5-HT2A selectivity; they exhibited features of 5-HTIA antagonists. Those results suggested that the mode of substitution of the terminal amide moiety in the tested tetramethylene arylpiperazines was not significant for their 5-HTIA receptor activity. Conformational analysis calculations indicated that despite its great capacity for adaptation at 5-HTIA receptor site, an aryl substituent in position 4 in the pyrid-2(1H)-one ring destabilized the ligand-5-HT1A receptor complex formation in the case of trimethylene derivatives. Diarylsubstituted derivatives (5a-8a and 5b-8b) were characterized by a low 5-HT2A affinity (Ki > 446 nM) regardless of the spacer length, while those with the tetramethylene aliphatic chain had a higher 5-HT2A affinity than the remaining investigated compounds.

Arylpiperazine derivatives of 3-propyl-beta-tetralonohydantoin as new 5-HT1A and 5-HT2A receptor ligands.

A series of new analogues of 3-[3-(4-arylpiperazinyl)-propyl]-cyclo-hexane-1',5-spirohydantoin (2), with aromatic ring fused in amide moiety (4-9) were synthesized and evaluated for affinity at 5-HTIA and 5-HT2A receptors. The influence of the substitution mode in the phenyl ring of phenylpiperazine moiety on the affinity for both receptors has been discussed. The most potent 5-HTIA (9, Ki = 53 nM) and 5-HT2A (4, 6, 8 and 9; Ki = 14-76 nM) ligands were evaluated in in vivo tests. The obtained results indicate that all in vivo tested compounds showed pharmacological profile of 5-HT2A antagonists. Additionally, a m-CF3 derivative (9), behaved like a partial agonist (agonist of pre- and antagonist of postsynaptic) of 5-HTIA receptors and may offer a new lead for the development of potential psychotropic agents.

Lack of effect of repeated treatment with a glycineB receptor partial agonist on the amphetamine-induced hyperactivity in rats.

The effect of acute and repeated (once daily, 14 days) administration of a potential antidepressant, the glycineB partial agonist 1-aminocyclopropanecarboxylic acid (ACPC, 100-400 mg/kg, ip), on the hyperactivity induced by amphetamine (0.5 mg/kg, sc) in rats was studied. Neither acute nor repeated treatment with the drug affected the hyperlocomotion induced by amphetamine. The obtained results indicate that ACPC does not resemble antidepressant drugs in this behavioral model.

9-substituted 1,2,3,4-tetrahydro-beta-carbolin-1-ones, new 5-HT1A and 5-HT2A receptor ligands.

Three series of new 9-substituted 1,2,3,4-tetrahydro-beta-carbolin-1-ones with 2-, 3- and 4-membered alkyl chain (1, 2 and 3, respectively) were synthesized, and the effect of some structural modifications on their 5-HT1A and 5-HT2A receptor affinities and functional in vivo properties was discussed. Radioligand binding measurements showed that the majority of compounds had a distinct affinity for 5-HT1A (1b, 2a, 2b, 2c, 3b; Ki = 0.3-64 nM) and 5-HT2A receptors (1b, 2b, 2c, 3b; Ki = 0.9-80 nM). The most potent 5-HT1A (1b, 2a, 2b, 3b) and 5-HT2A (1b, 2b, 3b) ligands were evaluated in in vivo tests. The obtained results indicate that 1,2,3,4-tetrahydro-beta-carbolin-1-ones containing 1-(o-methoxyphenyl)piperazine (1-3b) show pharmacological profile of 5-HT1A postsynaptic antagonists (with very weak agonistic component) and 5-HT2A antagonists, compound with 1,2,3,4-tetrahydroisoquinoline (2a) is a pure 5-HT1A postsynaptic antagonist. Summing up, the connection of 1,2,3,4-tetrahydro-beta-carbolin-1-one moiety through the 2-4-membered alkyl spacer with 1-(o-methoxyphenyl)-piperazine, which is present in a variety of 5-HT1A ligands, allowed us to obtain the compounds with high and equal affinity for 5-HT1A/5-HT2A receptors and the expected functional properties, i.e. distinct antagonistic and weak agonistic activity at 5-HT1A postsynaptic receptors and antagonistic at 5-HT2A ones.

Participation of opioid mechanism in the antinociceptive effects induced by oxaprotiline enantiomers in mice.

The purpose of the present study was to assess the activity of (+)-oxaprotiline [(+)-OXA] (a noradrenaline uptake inhibitor) and (-)-oxaprotiline [(-)-OXA] (with unknown mechanism of action) in two experimental models of pain in mice, a hot plate test and a writhing syndrome induced by phenylbenzoquinone (PHBQ), and to determine whether the opioidergic system may be engaged in their antinociceptive effects. Morphine was used as a reference drug. Administration of (+)-OXA (0.31-5 mg/kg) and (-)-OXA (20 mg/kg) produced a statistically significant elevation of the nociceptive threshold, measured by the increased latencies in the hot plate test. Moreover, (+)-OXA (0.62-5 mg/kg) and (-)-enantiomer (5-20 mg/kg) decreased the number of writhing episodes induced by PHBQ in mice, (+)-enantiomer being more effective than (-)-OXA in either test. In the hot plate test, the analgesic effect induced by (+)-OXA (0.31 mg/kg) or (-)-OXA (20 mg/kg) was abolished by naloxone (2 mg/kg), an opioid receptor antagonist. In the writhing test, naloxone (2 mg/kg) partially, but not significantly, reduced the antinociceptive responses induced by (+)-OXA (0.62 mg/kg) or (-)-OXA (5 mg/kg). The obtained results show that both OXA enantiomers produce antinociception in mice which can be, at least partially, connected with opioid system.

Structure-activity relationship studies of CNS agents. Part 38. Novel 1,4-benzoxazin-3(4H)-one, 1,2-benzoxazolin-3-one and 1,3-benzoxazolin-2,4-dione arylpiperazine derivatives with different 5-HT1A and antagonistic 5-HT2A activities.

New 1-arylpiperazine (series d-f) and 1,2,3,4-tetrahydroisoquinoline (series g) derivatives of 1,4-benzoxazin-3(4H)-one 1, 1,2-benzoxazolin-3-one 2, and 1,3-benzoxazolin-2,4-dione 3 with an n-butyl chain were synthesized in order to explore the effect of spacer elongation on their binding affinity and in vivo functional activity at 5-HT1A and 5-HT2A receptors in comparison with trimethylene analogues (a, bc). 5-HT1A receptor binding constants of derivatives 1d-g, 2d-f, and 3d-f were very high (Ki = 1.25-54 nM), and 5-HT2A affinities were maintained at a similar, high level (Ki = 27-85 nM) for series d and e, and moderate (Ki = 246-495 nM) for series f. In respect of a spacer, the obtained results showed either no effect or a slight increase in the 5-HT1A/5-HT2A affinity in case of derivatives of 1 and 2, respectively. A striking effect was observed for derivatives 3d and 3f, whose 5-HT1A affinity was reinforced by two orders of magnitude with a simultaneous decrease in 5-HT2A binding constants in comparison with trimethylene analogues. As shown by X-ray crystallography, this phenomenon may be attributed to the position of non-carbonyl oxygen atom in the amide moiety. In vivo studies demonstrated that compounds 1e-g, 2d-f, and 3f behaved like typical postsynaptic 5-HT1A receptor antagonists, whereas 3d and 3e might be qualified as their potential partial agonists. Moreover, 1e, 2e, and 3e demonstrated 5-HT2A receptor antagonistic properties. Of the tested compounds, two derivatives showed some very outstanding properties: 3e may be regarded as a potential anxiolytic and/or antidepressant agent, while 3f as a new potent 5-HT1A antagonist.

On the bioactive conformation of NAN-190 (1) and MP3022 (2), 5-HT(1A) receptor antagonists.

Structural modifications of 1, a postsynaptic 5-HT(1A) receptor antagonist, provided its flexible (8, 12) and rigid (7, 9, 11, 13) analogues. Compounds 7, 8, 9, and 11 showed high 5-HT(1A) receptor affinity (K(i) = 4-72 nM). They acted as 5-HT(1A) postsynaptic receptor antagonists, since, like 1, they inhibited the behavioral syndrome, i.e., flat body posture (FBP) and forepaw treading (FT), in reserpine-pretreated rats as well as the lower lip retraction (LLR) in rats, both induced by 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), a 5-HT(1A) receptor agonist. Compound 12, which demonstrated high 5-HT(1A) receptor affinity (K(i) = 50 nM), revealed properties of a partial 5-HT(1A) receptor agonist: it induced LLR and, at the same time, inhibited FT in rats. Compound 13 (K(i) = 1600 nM) was not tested in a behavioral study. Restriction of the conformational freedom in 2, a full 5-HT(1A) receptor antagonist, yielded compound 14 with high 5-HT(1A) receptor affinity (K(i) = 47 nM) and partial agonist properties at postsynaptic 5-HT(1A) receptors in the above tests in vivo; i.e., it induced LLR and inhibited FBP and FT in rats. New constrained analogues of 1 and 2 (compounds 7 and 14, respectively) were also synthesized to recognize a bioactive conformation of those 5-HT(1A) receptor antagonists. On the basis of in vitro and in vivo investigations, binding and functional properties of compound 7 were found to reflect those of 1 at 5-HT(1A) receptors. On the other hand, compound 14, a rigid analogue of 2, showed a different activity in vivo in comparison with the parent compound. PM3 and MM calculations revealed the existence of three low-energy conformers of 7 and six of 14, all of them belonging to the extended family of conformations. The optimized structures of both analogues had a different angle between aromatic planes of terminal fragments; moreover, the heteroaromatic system of those molecules occupied various space regions. Our present study provides support to the hypothesis that the bioactive conformation of 1, responsible for its postsynaptic 5-HT(1A) receptor antagonism, is an extended linear structure represented by 7.