Biotransformation of metenolone acetate and epiandrosterone by fungi and evaluation of resulting metabolites for aromatase inhibition.

The present study describes the microbial transformation of anabolic drugs, metenolone acetate (1), and epiandrosterone (6). Three new metabolites, 6ß,17ß-dihydroxy-1-methyl-3-oxo-5α-androst-1-en (2), 5α,15α-dihydroxy-1-methyl-3-oxo-1-en-17-yl acetate (3), 15ß-hydroxy-1-methyl-3-oxo-5α-androst-1,4-dien-17-yl acetate (4), and a known metabolite, 17ß-hydroxy-1-methyl-4-androstadiene-3-one (5) were obtained by biotransformation of metenolone acetate (1) via Trametes hirsuta mushroom. Metabolites 7, and 8 were obtained from the incubation of epiandrosterone (6) with Cunninghamella blakesleeana. While bioconversion of compound 6 with Aspergillus alliaceus yielded seven known metabolites 9-15. Modern spectroscopic techniques were employed for the structure elucidation of biotransformed products. All compounds were evaluated for their aromatase inhibitory activity. Among them, new metabolite 3 exhibited a significant human placental aromatase activity with an IC50 = 19.602 ± 0.47 µM, as compared to standard anti-cancer drug exemestane (IC50 = 0.232 ± 0.031 µM), whereas, metabolite 5 (IC50 = 0.0049 ± 0.0032 µM) exhibited a very potent activity. While substrate 6, and metabolites 2, 7, and 9 were found inactive. Aromatase plays a key role in the biosynthesis of estrogen hormone, responsible for cancer cell proliferation. Its inhibition is therefore targeted for the treatment of ER + breast cancer. Further structural modifications (lead optimization) of compound 3 can lead to more potent aromatase inhibition for possible treatment of ER + breast cancer.

Synthesis of 2-Aminopyrimidine Derivatives and Their Evaluation as ß-Glucuronidase Inhibitors: In Vitro and In Silico Studies.

Currently the discovery and development of potent ß-glucuronidase inhibitors is an active area of research due to the observation that increased activity of this enzyme is associated with many pathological conditions, such as colon cancer, renal diseases, and infections of the urinary tract. In this study, twenty-seven 2-aminopyrimidine derivatives 1-27 were synthesized by fusion of 2-amino-4,6-dichloropyrimidine with a variety of amines in the presence of triethylamine without using any solvent and catalyst, in good to excellent yields. All synthesized compounds were characterized by EI-MS, HREI-MS and NMR spectroscopy. Compounds 1-27 were then evaluated for their ß-glucuronidase inhibitory activity, and among them, compound 24 (IC50 = 2.8 ± 0.10 µM) showed an activity much superior to standard D-saccharic acid 1,4-lactone (IC50 = 45.75 ± 2.16 µM). To predict the binding mode of the substrate and ß-glucuronidase, in silico study was performed. Conclusively, this study has identified a potent ß-glucuronidase inhibitor that deserves to be further studied for the development of pharmaceutical products.

Ascaris induced acute pancreatitis in paediatric population - a case series.

The most common helminthic parasitic infection inhabiting human intestine is Ascaris lumbricoides (AL). Being the largest of the helminthic family, it infects almost one billion people worldwide, but any information about local population is unavailable especially in children. When patients present with abdominal pain, having ascaris induced pancreatitis never meets the differential diagnosis list even though AL itself is highly prevalent in our part of the world. Infected patients can present with a variety of symptoms depending on the location of parasite. If the biliary tree is inhabited, patients usually present with symptoms of choledocholithiasis or pancreatitis. We report the case series of 3 patients from paediatric age group, having acute pancreatitis secondary to AL. Patients had upper abdominal pain of varying duration. Ultrasound abdomen showed worm inside the Common Bile Duct (CBD) in all 3 patients. Endoscopic retrograde cholangio-pancreatography (ERCP) showed worms coming out of the ampullary orifice. Two patients received albendazole orally post ERCP and were discharged after complete resolution of symptoms with advice of repeat ERCP after 6 weeks, however one patient was advised Magnetic resonance cholangio-pancreatography (MRCP).

OCCURRENCE OF UNUSUAL HAEMOGLOBINOPATHIES IN BALOCHISTAN: HB SD AND HB SE - PRESENTATION WITH OSTEOMYELITIS.

OBJECTIVE: To describe two cases of unusual variants of sickle cell disease. CASE DESCRIPTION: We present two cases of sickle cell disease variants (haemoglobinopathies), from unrelated families, in the state of Balochistan (Pakistan). One was diagnosed with sickle cell disease in the haemoglobin electrophoresis, whereas the other was diagnosed with sickle cell SE disease. Both were diagnosed based on the presentation of osteomyelitis. COMMENTS: Haemoglobin SD disease (Hb SD) and haemoglobin SE disease (Hb SE) are rare haemoglobinopathies in the world. The lack of available literature suggests that both are variants of sickle cell disease (SCD), with heterogeneous nature. The prevalence of sickle cell disease with compound heterozygotes was found at a variable frequency in the population of the Asian Southeast. The frequency of osteomyelitis in SCD is 12 to 18%, but its occurrence among variant haemoglobinopathies is little reported. Both reported cases presented with osteomyelitis as a characteristic of the disease presentation.

Occurrence of unusual haemoglobinopathies in balochistan: hb sd and hb se - presentation with osteomyelitis / Ocorrência de hemoglobinopatias incomuns no baluchistão: hb sd e hb se - apresentação com osteomielite

ABSTRACT Objective: To describe two cases of unusual variants of sickle cell disease. Case description: We present two cases of sickle cell disease variants (haemoglobinopathies), from unrelated families, in the state of Balochistan (Pakistan). One was diagnosed with sickle cell disease in the haemoglobin electrophoresis, whereas the other was diagnosed with sickle cell SE disease. Both were diagnosed based on the presentation of osteomyelitis. Comments: Haemoglobin SD disease (Hb SD) and haemoglobin SE disease (Hb SE) are rare haemoglobinopathies in the world. The lack of available literature suggests that both are variants of sickle cell disease (SCD), with heterogeneous nature. The prevalence of sickle cell disease with compound heterozygotes was found at a variable frequency in the population of the Asian Southeast. The frequency of osteomyelitis in SCD is 12 to 18%, but its occurrence among variant haemoglobinopathies is little reported. Both reported cases presented with osteomyelitis as a characteristic of the disease presentation.

RESUMO Objetivo: Descrever dois casos de variantes raras da hemoglobinopatia falciforme. Descrição do caso: Apresentamos aqui dois casos de hemoglobinopatias variantes das células falciformes, de famílias não relacionadas, no estado do Baluchistão (Paquistão), sendo um diagnosticado como doença da hemoglobina SD na eletroforese de hemoglobina, enquanto o outro com doença da hemoglobina SE. Ambos foram diagnosticados a partir da apresentação de osteomielite. Comentários: Hemoglobina SD (Hb SD) e hemoglobina SE (Hb SE) são hemoglobinopatias raras no mundo. A escassez de literatura disponível sugere que ambas são variantes da doença falciforme (DF) com natureza heterogênea. A prevalência de hemoglobinopatia falciforme com heterozigosidade composta foi encontrada com frequência variável na população do sudeste asiático. A frequência de osteomielite na DF é de 12 a 18%, mas sua ocorrência entre as hemoglobinopatias falciformes variantes é pouco relatada. Os dois casos reportados apresentaram osteomielite como característica de apresentação da doença.

Bioinformatics: A rational combine approach used for the identification and in-vitro activity evaluation of potent ß-Glucuronidase inhibitors.

Identification of hotspot drug-receptor interactions through in-silico prediction methods (Pharmacophore mapping, virtual screening, 3DQSAR, etc), is considered as a key approach in drug designing and development process. In the current design study, advanced in-silico based computational techniques were used for the identification of lead-like molecules against the targeted receptor ß-glucuronidase. The binding pattern of a potent inhibitor in the ligand-receptor X-ray co-crystallize complex was used to identify and extract the structure-base Pharmacophore features. Based on these observations; five structure-based pharmacophore models were derived to conduct the virtual screening of ICCBS in-house data-base. Top-ranked identified Hits (33 compounds) were selected to subject for in-vitro biological activity evaluation against ß-glucuronidase enzyme; out of them, twenty compounds (61% of screened compounds) evaluated as actives, however eleven compounds were found to have significantly higher inhibitory activity, including compounds 1, 5-8, 10, 12-13, and 17-19 with IC50 values ranging from 1.2 µM to 34.9 µM. Out of the eleven potent inhibitors, seven compounds 1, 5, 6, 7, 8, 13, and 19 were found new, and evaluated first time for the ß-glucuronidase inhibitory activity. Compounds 1, 5 and 19 exhibited a highly potent inhibition in uM of ß-glucuronidase enzyme with non-cytotoxic behavior against the mouse fibroblast (3T3) cell line. Our combined in-silico and in-vitro results revealed that the binding pattern analysis of the eleven potent inhibitors, showed almost similar non-covalent interactions, as observed in case of our validated pharmacophore model. The obtained results thus demonstrated that the virtual screening minimizes false positives, and provide a template for the identification and development of new and more potent ß-glucuronidase inhibitors with non-toxic effects.

2-Oxo-1,2,3,4-tetrahydropyrimidines Ethyl Esters as Potent ß- Glucuronidase Inhibitors: One-pot Synthesis, In vitro and In silico Studies.

BACKGROUND: Glucuronidation is essential for the metabolism and excretion of toxic substances. ß-Glucuronidase enzyme slows down the process of glucuronidation, and thus plays an important role in the on-set of colorectal carcinoma, and many other diseases. Inhibition of ß- glucuronidase activity is thus identified as an important approach for the treatment of several diseases. OBJECTIVE: Current study was aimed to synthesize a library of 2-oxo-1,2,3,4-tetrahydropyrimidine and to evaluate their ß-glucuronidase inhibitory activity, and their mode of enzyme inhibition. METHOD: We synthesized a series of 2-oxo-1,2,3,4-tetrahydropyrimidines 1-25 by fusing urea, ethyl acetoacetate, and a variety of aldehydes using copper nitrate trihydrate as catalyst. All synthesized compounds were evaluated for their in vitro ß-glucuronidase inhibitory activity. In addition, molecular docking studies were also performed by using MOE docking tools. RESULTS: Eighteen compounds showed inhibitory activity better than the standard D-saccharic acid 1,4-lactone, a well known ß-glucuronidase inhibitor (IC50 = 45.75 ± 2.16 µM). Compound 20 (IC50 = 1.36 ± 0.03 µM) showed an excellent inhibitory activity, thirty-five folds superior to the standard. Docking results highlighted the role of various chemical moieties at different positions on 2- oxo-1,2,3,4-tetrahydropyrimidine skeleton in enzyme inhibitory activity. CONCLUSION: This study has identified a class of potent ß-glucuronidase inhibitors with the potential to be investigated further.

Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity evaluation of its metabolites.

Seven metabolites were obtained from the microbial transformation of anabolic-androgenic steroid mibolerone (1) with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina. Their structures were determined as 10ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (2), 6ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (3), 6ß,10ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (4), 11ß,17ß-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one (5), 1α,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (6), 1α,11ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (7), and 11ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (8), on the basis of spectroscopic studies. All metabolites, except 8, were identified as new compounds. This study indicates that C. blakesleeana, and C. echinulata are able to catalyze hydroxylation at allylic positions, while M. phaseolina can catalyze hydroxylation of CH2 and CH3 groups of substrate 1. Mibolerone (1) was found to be a moderate inhibitor of ß-glucuronidase enzyme (IC50 = 42.98 ± 1.24 µM) during random biological screening, while its metabolites 2-4, and 8 were found to be inactive. Mibolerone (1) was also found to be significantly active against Leishmania major promastigotes (IC50 = 29.64 ± 0.88 µM). Its transformed products 3 (IC50 = 79.09 ± 0.06 µM), and 8 (IC50 = 70.09 ± 0.05 µM) showed a weak leishmanicidal activity, while 2 and 4 were found to be inactive. In addition, substrate 1 (IC50 = 35.7 ± 4.46 µM), and its metabolite 8 (IC50 = 34.16 ± 5.3 µM) exhibited potent cytotoxicity against HeLa cancer cell line (human cervical carcinoma). Metabolite 2 (IC50 = 46.5 ± 5.4 µM) also showed a significant cytotoxicity, while 3 (IC50 = 107.8 ± 4.0 µM) and 4 (IC50 = 152.5 ± 2.15 µM) showed weak cytotoxicity against HeLa cancer cell line. Compound 1 (IC50 = 46.3 ± 11.7 µM), and its transformed products 2 (IC50 = 43.3 ± 7.7 µM), 3 (IC50 = 65.6 ± 2.5 µM), and 4 (IC50 = 89.4 ± 2.7 µM) were also found to be moderately toxic to 3T3 cell line (mouse fibroblast). Interestingly, metabolite 8 showed no cytotoxicity against 3T3 cell line. Compounds 1-4, and 8 were also evaluated for inhibition of tyrosinase, carbonic anhydrase, and α-glucosidase enzymes, and all were found to be inactive.

Synthesis of pyrimidine-2,4,6-trione derivatives: Anti-oxidant, anti-cancer, α-glucosidase, ß-glucuronidase inhibition and their molecular docking studies.

This paper describes a facile protocol, efficient, and environmentally benign for the synthesis a series of barbiturate acid substituted at C5 position 3a-o. The desired compounds subjected in vitro for different set of bioassays including against anti-oxidant (DPPH and super oxide scavenger assays), anti-cancer, α-glucosidase and ß-glucuronidase inhibitions. Compound 3m (IC50=22.9±0.5µM) found to be potent α-glucosidase enzyme inhibitors and showed more activity than standard acarbose (IC50=841±1.73µM). Compound 3f (IC50=86.9±4.33µM) found to be moderate ß-Glucuronidase enzyme inhibitors and showed activity comparatively less than the standard d-saccharic acid 1,4-lactone (IC50=45.75±2.16µM). Furthermore, in sillico investigation was carried out to investigate bonding mode of barbiturate acid derivatives.

Synthesis and ß-glucuronidase inhibitory activity of 2-arylquinazolin-4(3H)-ones.

2-Arylquinazolin-4(3H)-ones 1-25 were synthesized by reacting anthranilamide with various benzaldehydes using CuCl2·2H2O as a catalyst in ethanol under reflux. Synthetic 2-arylquinazolin-4(3H)-ones 1-25 were evaluated for their ß-glucuronidase inhibitory potential. A trend of inhibition IC50 against the enzyme in the range of 0.6-198.2µM, was observed and compared with the standard d-saccharic acid 1,4-lactone (IC50=45.75±2.16µM). Compounds 13, 19, 4, 12, 14, 22, 23, 25, 15, 8, 17, 11, 21, 1, 3, 18, 9, 2, and 24 with the IC50 values within the range of 0.6-44.0µM, indicated that the compounds have superior activity than the standard. The compounds showed no cytotoxic effects against PC-3 cells. A structure-activity relationship is established.

ß-glucuronidase inhibitory studies on coumarin derivatives.

Twenty-three (23) derivatives of coumarin (5-27) were synthesized and screened for their in vitro ß- glucuronidase (E. coli) inhibitory activities. Only three compounds, 7,8-dihydroxy-4-methyl-2H-chromen-2-one (9) (IC50 = 52.39 ± 1.85 µM), 3-chloro-6-hexyl-7-hydroxy-4-methyl-2H-chromen-2-one (18) (IC50 = 60.50 ± 0.87 µM), and 3,6- dichloro-7-hydroxy-4-methyl-2H-chromen-2-one (15) (IC50 = 380.26 ± 0.92 µM) displayed activities against ß- glucuronidase as compared to standard D-saccharic acid 1,4-lactone (IC50 = 45.75 ± 2.16 µM). The results indicated that the activity of the synthetic coumarins depends upon the substituents present on the coumarin skeleton.