Pyrazolidine-3,5-dione derivatives as potent non-steroidal agonists of farnesoid X receptor: virtual screening, synthesis, and biological evaluation

The identification of a novel pyrazolidine-3,5-dione based scaffold hit compound as Farnesoid X receptor (FXR) partial or full agonist has been accomplished by means of virtual screening techniques. A series of pyrazolidine-3,5-dione derivatives (1a-u and 7) was designed, synthesized, and evaluated by a cell-based luciferase transactivation assay for their agonistic activities against FXR. Most of them showed agonistic potencies and 10 of them (1a, 1b, 1d-f, 1j, 1n, 1t, 5b, and 7) exhibited lower EC(50) values than the reference drug CDCA. Molecular modeling studies for the representative compounds 1a, 1d, 1f, 1j, 1n, 1u, 5b, and 7 were also presented. The novel structural scaffold has provided a new direction for finding potent and selective FXR partial and full agonists (referred to as 'selective bile acid receptor modulators', SBARMs).     

Synthesis and evaluation of biological and antitumor activities of 5,7-dimethyl- oxazolo[5,4-d]pyrimidine-4,6(5H,7H)-dione derivatives as novel inhibitors of FGFR1

Abstract

A series of 5,7-dimethyl-oxazolo[5,4-d]pyrimidine-4,6(5H,7H)-dione derivatives, N5a–5l, was designed, synthesized and evaluated for their FGFR1-inhibition ability as well as cytotoxicity against three cancer cell lines (H460, B16F10 and A549) in vitro. Several compounds displayed good-to-excellent potency against these cancer cell lines compared to SU5402. Structure–activity relationship analyses indicated that compounds with a rigid structure and more heteroatoms at the side chain of the parent ring were more effective than those without these substitutions. The compound N5g (37.4% FGFR1 inhibition at 1.0?μM) was identified to have the most potent antitumor activities, with IC₅₀ values of 5.472, 4.260 and 5.837?μM against H460, B16F10 and A549 cell lines, respectively. Together, our results suggest that 5,7-dimethyl-oxazolo[5,4-d]pyrimidine-4,6(5H,7H)-dione derivatives may serve as potential agents for the treatment of FGFR1-mediated cancers.     

The degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590 under anaerobic conditions.

The bacterial degradation of cholic acid under anaerobic conditions by Pseudomonas sp. N.C.I.B. 10590 was studied. The major unsaturated neutral compound was identified as 12 beta-hydroxyandrosta-4,6-diene-3,17-dione, and the major unsaturated acidic metabolite was identified as 12 alpha-hydroxy-3-oxochola-4,6-dien-24-oic acid. Eight minor unsaturated metabolites were isolated and evidence is given for the following structures: 12 alpha-hydroxyandrosta-4,6-diene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-4,6-dien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 alpha-hydroxyandrosta-1,4-diene-3,17-dione, 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione, 3,12-dioxochola-4,6-dien-24-oic acid and 12 alpha-hydroxy-3-oxopregna-4,6-diene-20-carboxylic acid. In addition, a major saturated neutral compound was isolated and identified as 3 beta,12 beta-dihydroxy-5 beta-androstan-17-one, and the only saturated acidic metabolite was 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholan-24-oic acid. Nine minor saturated neutral compounds were also isolated, and evidence is presented for the following structures: 12 beta-hydroxy-5 beta-androstane-3,17-dione, 12 alpha-hydroxy-5 beta-androstane-3,17-dione, 3 beta,12 alpha-dihydroxy-5 beta-androstan-17-one, 3 alpha,12 beta-androstan-17-one, 3 alpha,12 alpha-dihydroxy-5 beta-androstan-17-one, 5 beta-androstane-3 beta,12 beta,17 beta-triol, 5 beta-androstane-3 beta,12 alpha,17 beta-triol, 5 beta-androstane-3 alpha,12 beta,17 beta-triol and 5 beta-androstane-3 alpha,12 alpha,17 beta-triol. The induction of 7 alpha-dehydroxylase and 12 alpha-dehydroxylase enzymes is discussed, together with the significance of dehydrogenation and ring fission under anaerobic conditions.     

Both 5-arylidene-2-thioxodihydropyrimidine-4,6(1H,5H)-diones and 3-thioxo-2,3-dihydro-1H-imidazo[1,5-a]indol-1-ones are light-dependent tumor necrosis factor-alpha antagonists

Based on the realization that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones are tumor necrosis factor-alpha antagonists, we discovered two additional classes of antagonists: 3-thioxo-2,3-dihydro-1H-imidazo[1,5-a]indol-1-ones (via rational design) and 5-arylidene-2-thioxodihydropyrimidine-4,6(1H,5H)-diones (via computer-guided screening). Chemical modification of the lead structures showed that the structure-activity relationship profiles for both of these series were dependent on the electronic properties of the molecules. Subsequent studies showed that they were light-dependent inhibitors.     

Aza analogues of thalidomide: synthesis and evaluation as inhibitors of tumor necrosis factor-alpha production in vitro

A synthetic entry to derivatives of the new classes of 5-phthalimidouracils and 5-phthalimidobarbituric acids is reported. These 5-phthalimidopyrimidines as well as phthalimido-2,4-difluorobenzenes were designed as analogues of thalidomide, a well known inhibitor of TNF-alpha production. A preliminary in vitro investigation of the compounds as inhibitors of the TNF-alpha production was performed. Among the compounds of the present series, 5-ethyl-1-phenyl-5-(tetrafluorophthalimido)barbituric acid and 2-(2,4-difluorophenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione were proved to be potent inhibitors. Both compounds showed inhibitory activity in the lower micromolar range on the LPS-induced TNF-alpha production in human monocytes.     

Synthesis of benzamide derivatives as TRPV1 antagonists

From hit compounds identified by high throughput screening (HTS), we have found compound 1 as a lead TRPV1 antagonist and confirmed its potential as a treatment for pain. Compound 1 has led to potent TRPV1 antagonistic benzamide derivatives ((+/-)-2: human IC(50)=23 nM, (+/-)-3: human IC(50)=14 nM in the capsaicin-induced calcium influx assay) containing indole and naphthyl moieties, obtained by elaboration of the tryptamine scaffold or via bioisosteric replacements.     

Structure-activity relationships of progesterone derivatives that bind to the digitalis receptor: modifications in A and B rings

A number of progesterone derivatives, having a 17 alpha-acetoxy group and various functions at C-3 and C-6, interact at the cardiac glycoside (CG) binding site, using [3H]ouabain in a radioligand binding assay (RBA) with membranes from dog myocardium. We now report on results of structure-activity studies concerned with modification of the A and B rings as they influence potency in the RBA. Some progesterone derivatives with 5 alpha- or 5 beta-stereochemistry show weak receptor competing activity. Among the congeners highest potency is associated with the presence of C-4 or C-4,6 unsaturation and a C-6 substituent (CH3, Cl, Br) whose importance appears to reside in its steric rather than electronic character. The C-3 function may be carbonyl, 3 beta-hydroxy or 3 beta-acetoxy when associated with C-4 or C-4,6 unsaturation. In compounds with other substituents that promote activity, C-6 alpha substitution with -CH3, -Cl, or -Br strongly enhances activity; -F, -OCH3, carbonyl, or the unsubstituted compound promotes weak binding; and -OC2H5, -OAc, -OCOOCH3, or -OH eliminates binding activity. Receptor interaction with the double bond at C-4, but not C-5, appears to be particularly important for binding. The most potent analog identified thus far is chlormadinone acetate (17 alpha-acetoxy-6-chloropregna-4,6-diene-3,20-dione), which has 1/20 the potency of ouabain in the RBA. Studies to determine optimal structural requirements for CG-receptor binding by these hormonal steroid congeners, in conjunction with appropriate biological assays, may provide insight into the nature of a putative endogenous counterpart, lead to a better understanding of the mode of action of the CG and yield CG-like compounds with superior therapeutic properties.     

New pyrrolopyrimidin-6-yl benzenesulfonamides: potent A2B adenosine receptor antagonists

A new series of 4-(1,3-dialkyl-2,4-dioxo-2,3,4,5-tetrahydro-1H-pyrrolo[3,2-d]pyrimidin-6-yl)benzenesulfonamides has been identified as potent A2B adenosine receptor antagonists. The products have been evaluated for their binding affinities for the human A2B, A1 and A3 adenosine receptors. 6-(4-{[4-(4-Bromobenzyl)piperazin-1-yl]sulfonyl}phenyl)-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-dione (16) showed a high affinity for the A2B adenosine receptor (IC50=1 nM) and selectivity (A1: 183x; A3: 12660x). Synthesis and SAR of this novel class of compounds showing improved absorption properties is presented herein.     

Synthesis of 5-benzyl-2-phenylpyrazolo[1,5-a]pyrazin-4,6(5H,7H)-dione derivatives and discovery of an apoptosis inducer for H322 lung cancer cells

A series of substituted 5-benzyl-2-phenylpyrazolo[1,5-a]pyrazin-4,6(5H,7H)-dione derivatives was synthesized by one-step reaction of ethyl 3-phenyl-1H-pyrazole-5-carboxylate derivatives and N-arylalkyl-2-chloroacetamide. Structures of the compounds were determined by IR, (1)H NMR and mass spectroscopy. In addition, a representative single-crystal structure was characterized by using X-ray diffraction analysis. The compound 5j could selectively inhibit the growth of H322 lung cancer cells which contain a mutated p53 gene in a dose-dependent manner through inducing apoptosis of cells.     

Hydroxylation of testosterone at carbons 1, 2, 6, 7, 15 and 16 by the hepatic microsomal fraction from adult female C57BL/6J mice.

The metabolism of a mixture of [4-14C]- and [7 beta-2H]testosterone by the hepatic microsomal fraction from adult femal C57BL/6J mice has been investigated. The following metabolites were identified by their mass spectra and by their retention times on gas chromatography on one or two phases: 1epsilon-, 2beta-, 6alpha-, 6beta-, 7alpha-, 15alpha-, 15beta-, 16alpha- and 16beta-hydroxytestosterone; 6alpha-, 6beta- and 7alpha-hydroxy-4-androstene-3,17-dione; and 4-androstene-3,17-dione. A compound tentatively identified as 6- or 7-oxotestosterone was also isolated. 17beta-Hydroxy-4,6-androstadien-3-one, 17beta-hydroxy-1,4-androstadien-3-one and 4,6-androstadiene-3,17-dione were identified but are considered to arise non-enzymatically from 7alpha-hydroxytestosterone, 1epsilon-hydroxytestosterone and 7alpha-hydroxy-4-androstene-3,17-dione, respectively.     

Studies on the endocrine properties of a new progestational steroid 4,6-dichloro-16-methylene-17-hydroxy-4,6-pregnadiene-3,20-dione 17-acetate (MDAP)

MDAP (4,6-dichloro-16-methylene-17-hydroxy-4,6-pregnadiene-3,20-dione 17-acetate) was found to be approximately 100 times more potent than progesterone and 5 to 10 times more potent than chlormadinone acetate (6-chloro-17-hydroxy-4,6-pregnadiene-3, 20-dione 17-acetate) in the Clauberg-McPhail assay. MDAP manifested significant antigonadotropic and anti-androgenic activities and caused adrenal atrophy. The compound was also weakly uterotropic, thymolytic, and anti-estrogenic.     

Photochemical synthesis and anticancer activity of barbituric acid,thiobarbituric acid,thiosemicarbazide, and isoniazid linked to 2-phenyl indole derivatives

2-Phenyl-1H-indole-3-carbaldehyde-based barbituric acid, thiobarbituric acid, thiosemicarbazide, isoniazid, and malononitrile derivatives were synthesized under photochemical conditions. The antitumor activities of the synthesized compounds were evaluated on three different human cancer cell lines representing prostate cancer cell line DU145, Dwivedi (DWD) cancer cell lines, and breast cancer cell line MCF7. All the screened compounds possessed moderate anticancer activity, and out of all the screened compounds, 5-{1[2-(4-chloro-phenyl)2-oxo-ethyl]-2-phenyl-1H-indole-3-ylmethylene}-2-thioxo-dihydro-pyrimidine-4,6-dione (2b) and 5-{1[2-(4-methoxy-phenyl)2-oxo-ethyl]-2-phenyl-1H-indole-3-ylmethylene}-2-thioxo-dihydro-pyrimidine-4,6-dione (2d) exhibited marked antitumor activity against used cell lines. Additionally, barbituric acid derivatives were selective to inhibit cell line DWD and breast cancer cell lines.     

Synthesis and pharmacology of pyrido[2,3-d]pyrimidinediones bearing polar substituents as adenosine receptor antagonists

Amino-substituted pyrido[2,3-d]pyrimidinediones have previously been found to bind to adenosine A1 and A2A receptors in micromolar concentrations. The present study was aimed at studying the structure-activity relationships of this class of compounds in more detail. Most of the investigated compounds were provided with polar substituents, such as ethoxycarbonyl groups and basic amino functions, in order to improve their water-solubility. The compounds were synthesized starting from 6-amino-1,3-dimethyluracil via different reaction sequences involving (cyano)acetylation, Vilsmeier formylation, or reaction with diethyl ethoxymethylenemalonate (EMME). The most potent and selective compound of the present series was 6-carbethoxy-1,2,3,4-tetrahydro-1,3-dimethyl-5-(2-naphthylmethyl)aminopyrido[2,3-d]pyrimidine-2,4-dione (11c) with a Ki value of 5 nM at rat and 25 nM at human A1 receptors. The compound was more than 60-fold selective versus A3 and more than 300-fold selective versus A2A receptors. It showed an over 300-fold improvement with respect to the lead compound. In GTPgammaS binding studies at membranes of Chinese hamster ovary cells recombinantly expressing the human adenosine A1 receptor, 11c behaved as an antagonist with inverse agonistic activity. A regioisomer of 11c, 6-carbethoxy-1,2,3,4-tetrahydro-1,3-dimethyl-7-(2- naphthylmethyl)aminopyrido[2,3-d]pyrimidine-2,4-dione (7a) in which the 2-naphthylmethylamino substituent at position 5 of 11c was moved to the 7-position, was a relatively potent (Ki=226 nM) and selective (>20-fold) A3 ligand. In the series of compounds lacking an electron-withdrawing ethoxycarbonyl or cyano substituent in the 6-position, compounds with high affinity for adenosine A2A receptors were identified, such as 1,2,3,4-tetrahydro-1,3-dimethyl-5-(1-naphthyl)aminopyrido[2,3-d]pyrimidine-2,4-dione 16b (Ki human A2A=81.3 nM, Ki human A1=153 nM, and Ki human A3>10,000 nM).     

New thiazole carboxamides as potent inhibitors of Akt kinases

A new series of 2-substituted thiazole carboxamides were identified as potent pan inhibitors against all three isoforms of Akt (Akt1, Akt2 and Akt3) by systematic optimization of weak screening hit N-(1-amino-3-phenylpropan-2-yl)-2-phenylthiazole-5-carboxamide (1). One of the most potent compounds, 5m, inhibited the kinase activities of Akt1, Akt2 and Akt3 with IC(50) values of 25, 196 and 24nM, respectively. The compound also potently inhibited the phosphorylation of downstream MDM2 and GSK3β proteins, and displayed strongly antiproliferative activity in prostate cancer cells. The inhibitors might serve as lead compounds for further development of novel effective anticancer agents.     

SAR and evaluation of novel 5H-benzo[c][1,8]naphthyridin-6-one analogs as Aurora kinase inhibitors

Several potent Aurora kinase inhibitors derived from 5H-benzo[c][1,8]naphthyridin-6-one scaffold were identified. A crystal structure of Aurora kinase A in complex with an initial hit revealed a binding mode of the inhibitor within the ATP binding site and provided insight for structure-guided compound optimization. Subsequent SAR campaign provided a potent and selective pan Aurora inhibitor, which demonstrated potent target modulation and antiproliferative effects in the pancreatic cell line, MIAPaCa-2. Furthermore, this compound inhibited phosphorylation of histone H3 (pHH3) in mouse bone morrow upon oral administration, which is consistent with inhibition of Aurora kinase B activity.     

Discovery of a new insecticide lead by optimizing a target-diverse scaffold: tetrazolinone derivatives

In order to discover lead compounds with novel action mechanism, a series of tetrazolinone derivatives bearing structurally diverse substituents, 1-aryl-4-substituted-1,4-di-hydro-5H-tetrazol-5-ones 2, 1-((5-(alkylthio)-1,3,4-oxadiazol-2-yl)methyl)-4-(substituted)- phenyl-1H-tetrazol-5(4H)-ones 5, and 1-((5-(alkylthio)-1,3,4-thiadiazol-2-yl)methyl)-4- (substituted)phenyl-1H-tetrazol-5(4H)-ones 7, were designed and synthesized in good yields by a multiple-step synthetic procedure. The results of greenhouse in vivo test indicated that all the target compounds did not displayed herbicidal activity, however, some of them exhibited excellent in vivo insecticidal activity against Tetranychus cinnabarinus at the concentration of 250 mg L-1. To our knowledge, this is the first report about the insecticidal activity of tetrazolinone derivatives, which indicated that the tetrazolinone scaffold could be identified as a novel insecticidal lead structure. The present work demonstrated that optimizing a target-diverse scaffold is an effective way to discover new lead compounds with new action mechanism or biological activity.     

Design, synthesis, and pharmacological evaluation of new neuroactive pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives with in vivo hypnotic and analgesic profile

The present study describes the synthesis and pharmacological profiles of four novel pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives 2-5, which were structurally designed by using the sedative and analgesic drug zolpidem 1 as lead compound. The heterotricyclic system present in the target compounds 2-5 was constructed in good yields, exploiting a regioselective hetero Diels-Alder reaction of the key azabutadiene derivative 7 and functionalized N-phenylmaleimides 9-12. Additionally, we identified that 1-methyl-7-(4-nitrophenyl)-3-phenyl-3,6,7,8-tetrahydropyrazolo[3,4-b]pyrrolo[3,4-d]pyridine-6,8-dione derivative (LASSBio-873, 5) presented not only the most potent ability to promote sedation, which was similar to that induced by the standard benzodiazepine drug midazolam, but also potent central antinociceptive effect.     

2-Aryladenine derivatives as a potent scaffold for A1, A3 and dual A1/A3 adenosine receptor antagonists: Synthesis and structure-activity relationships

From a collection containing more than 1500 academic compounds, in silico screening identified a hit for the human A₁ adenosine receptor containing a new purine scaffold. To study the structure activity relationships of this new chemical series for adenosine receptors, a library of 24 purines was synthesized and tested in radioligand binding assays at human A₁, A_2A, A_2B and A₃ adenosine receptor subtypes. Fourteen molecules showed potent antagonism at A₁, A₃ or dual A₁/A₃ adenosine receptors. This purine scaffold is an important source for novel biochemical tools and/or therapeutic drugs.     

Synthesis and structure-activity relationships for biphenyl H3 receptor antagonists with moderate anti-cholinesterase activity

The combination of antagonism at histamine H(3) receptors and inhibition of acetylcholinesterase has been recently proposed as an approach to devise putative new therapeutic agents for cognitive diseases. The 4,4'-biphenyl fragment has been reported by us as a rigid scaffold leading to potent and selective non-imidazole H(3)-antagonists. Starting from these premises, the current work presents an expanded series of histamine H(3) receptor antagonists, characterized by a central 4,4'-biphenyl scaffold, where the structure-activity profile of both mono-basic and di-basic compounds is further explored and their ability to inhibit rat brain cholinesterase activity is determined. The steric properties and basicity of the terminal groups were modulated in symmetrical compounds, carrying identical substituents, and in asymmetrical compounds, having a piperidine ring at one end and different groups at the other. The length of the linker connecting the biphenyl scaffold to the terminal groups was also modulated. Binding studies at rat and human H(3) receptors evidenced the highest binding affinities for di-basic compounds, in the order of nM concentrations, and that the steric requirements for the two terminal groups are different. Many potent compounds showed good selectivity profiles over the other histamine receptors. Interestingly, some derivatives displayed a moderate ability to inhibit rat brain cholinesterase, for example compound 12 (1-[2-(4'-piperidinomethyl-biphenyl-4-yl)ethyl]piperidine) has a pIC(50)=5.96 for cholinesterase inhibition and high H(3) receptor binding affinity and antagonist potency (pK(i)=8.70; pK(B)=9.28). These compounds can be considered as rigid analogs of a recently reported class of dual-acting compounds and as a promising starting point for the design of new H(3)-antagonists with anti-cholinesterase activity.     

Synthesis and antituberculosis activity of 5-methyl/trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazone derivatives

New series of 5-methyl/trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazones 3a-t, 1-methyl-5-methyl/trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazones 4a-y and 5-trifluoromethoxy-1-morpholinomethyl-1H-indole-2,3-dione 3-thiosemicarbazones 5a-m were synthesized. The structures of the synthesized compounds were confirmed by spectral data and elemental analysis. The new 5-methyl/trifluoromethoxy-1H-indole-2,3-dione derivatives, along with previously synthesized 5-methyl-1H-indole-2,3-dione 3-thiosemicarbazones 6a-l, were evaluated for in vitro antituberculosis activity against Mycobacterium tuberculosis H37Rv. 5-Methyl-1H-indole-2,3-dione 3-thiosemicarbazones (3b, 3d, 3f, 6c, 6d, and 6f), 5-trifluoromethoxy-1H-indole-2,3-dione 3-thiosemicarbazones (3q-s) and 5-trifluoromethoxy-1-morpholinomethyl-1H-indole-2,3-dione 3-thiosemicarbazones (5e and 5j-l) were found to be the most potent inhibitors of M. tuberculosis growth described in this study.