17β-Arylsulfonamides of 17β-aminoestra-1,3,5(10)-trien-3-ol as highly potent inhibitors of steroid sulfatase

Steroid sulfatase (STS) catalyzes the desulfation of biologically inactive sulfated steroids to yield biologically active desulfated steroids and is currently being examined as a target for therapeutic intervention for the treatment of breast and other steroid-dependent cancers. Here we report the synthesis of a series of 17β-arylsulfonamides of 17β-aminoestra-1,3,5(10)-trien-3-ol and their evaluation as inhibitors of STS. Some of these compounds are among the most potent reversible STS inhibitors reported to date. Introducing n-alkyl groups into the 4'-position of the 17β-benzenesulfonamide derivative resulted in an increase in potency with the n-butyl derivative exhibiting the best potency with an IC(50) of 26 nM. A further increase in carbon units (to n-pentyl) resulted in a decrease in potency. Branching of the 4'-n-propyl group resulted in a decrease in potency while branching of the 4'-n-butyl group (to a tert-butyl group) resulted in a slight increase in potency (IC(50)=18 nM). Studies with 3'- and 4'-substituted substituted 17β-benzenesulfonamides with small electron donating and electron withdrawing groups revealed the 3'-bromo and 3'-trifluoromethyl derivatives to be excellent inhibitors with IC(50)'s of 30 and 23 nM, respectively. The 17β-2'-naphthalenesulfonamide was also an excellent inhibitor (IC(50)=20 nM) while the 17β-4'-phenylbenzenesulfonamide derivative was the most potent inhibitor of all the compounds studied with an IC(50) of 9 nM.     

2,4-disubstituted pyrimidines: a novel class of KDR kinase inhibitors

2,4-Disubstituted pyrimidines were synthesized as a novel class of KDR kinase inhibitors. Evaluation of the SAR of the screening lead compound 1 (KDR IC(50)=105 nM, Cell IC(50)=8% inhibition at 500 nM) led to the potent 3,5-dimethylaniline derivative 2d (KDR IC(50)=6 nM, cell IC(50)=19 nM).     

Synthesis, biochemical evaluation of a range of potent 4-substituted phenyl alkyl imidazole-based inhibitors of the enzyme complex 17alpha-Hydroxylase/17,20-Lyase (P45017alpha)

We report the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a number of azole-based compounds as inhibitors of the two components of the cytochrome P-450 enzyme 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), i.e. 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results suggest that the compounds synthesised are potent inhibitors, with 7-phenyl heptyl imidazole (11) (IC(50)=320 nM against 17alpha-OHase and IC(50)=100 nM against lyase); 1-[7-(4-fluorophenyl) heptyl] imidazole (14) (IC(50)=170 nM against 17alpha-OHase and IC(50)=57 nM against lyase); 1-[5-(4-bromophenyl) pentyl] imidazole (19) (IC(50)=500 nM against 17alpha-OHase and IC(50)=58 nM against lyase) being the most potent inhibitors within the current study, in comparison to ketoconazole (KTZ) (IC(50)=3.76 microM against 17alpha-OHase and IC(50)=1.66 microM against lyase). Furthermore, consideration of the inhibitory activity against the two components shows that all of the compounds tested are less potent towards the 17alpha-OHase in comparison to the lyase component, a desirable property in the development of novel inhibitors of P450(17alpha). From the modelling of these compounds onto the novel substrate heme complex (SHC) for the overall enzyme complex, the length of the compound, along with its ability to undergo interaction with the active site corresponding to the C(3) area of the steroidal backbone, are suggested to play a key role in determining the overall inhibitory activity.     

Design and synthesis of piperidine farnesyltransferase inhibitors with reduced glucuronidation potential

The design and synthesis of a novel piperidine series of farnesyltransferase (FTase) inhibitors with reduced potential for metabolic glucuronidation are described. The various substitution and exchange of the phenyl group at the C-2 position of the previously described 2-(4-hydroxy)phenyl-3-nitropiperidine 1a (FTase IC(50)=5.4nM) resulted in metabolically stable compounds with potent FTase inhibition (14a IC(50)=4.3nM, 20a IC(50)=3.0nM, and 50a IC(50)=16nM). Molecular modeling studies of these compounds complexed with FTase and farnesyl pyrophosphate are also described.     

Design and synthesis of xanthine analogues as potent and selective PDE5 inhibitors

We have discovered potent and selective xanthine PDE5 inhibitors. Compound 25 (PDE5 IC(50)=0.6 nM, PDE6/PDE5=101) demonstrated similar functional efficacy and PK profile to Sildenafil (PDE5 IC(50)=3.5 nM, PDE6/PDE5=7).     

Inhibition of carbonic anhydrase II by steroidal and non-steroidal sulphamates

Carbonic anhydrases (CAs) are expressed by many solid tumours where they may act to confer a growth advantage on malignant tissues. In this study we have examined the ability of a series of steroidal and non-steroidal sulphamates (originally developed as steroid sulphatase inhibitors) and related compounds to inhibit human CAII (hCAII) activity in vitro. Using a 96-well plate assay, oestrone-3-O-sulphamate (EMATE) and two coumarin-based sulphamate drugs (667 COUMATE and STX 118) were found to have IC(50) values of 25-59 nM for the inhibition of hCAII activity. These compounds therefore have a similar CAII inhibitory potency to that of acetazolamide (IC(50)=25 nM), a known hCAII inhibitor. Docking studies have been performed with selected compounds to the crystal structure of hCAII and excellent correlation of scores with biological activity was observed. This agrees with our recent observations when we were the first to report the inhibition of hCAII by STS inhibitors. These studies and initial results with docking to the crystal structure of the extracellular domain of hCAXII indicate that the STS sulphamate ester inhibitors should also be interesting candidates to pursue as inhibitors of CA isozymes that are over-expressed in human tumours.     

Synthesis and structure-activity relationships of novel IKK-beta inhibitors. Part 3: Orally active anti-inflammatory agents

A series of 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl)pyridine derivatives was synthesized and evaluated as I kappaB kinase beta (IKK-beta) inhibitors. Modification of a novel IKK-beta inhibitor 1 (IKK-beta IC(50)=1500 nM, Cell IC(50)=8000 nM) at the 4-phenyl ring and 6-phenol group on the pyridine core ring resulted in a marked increased in biological activities. An optimized compound, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile, exhibited excellent in vitro profiles (IKK-beta IC(50)=8.5 nM, Cell IC(50)=60 nM) and a strong oral efficacy in in vivo anti-inflammatory assays (significant effects at 1mg/kg, po in arachidonic acid-induced ear edema model in mice).     

Optimization of 1,4-diazepan-2-one containing dipeptidyl peptidase IV inhibitors for the treatment of type 2 diabetes

Following the discovery of N-acyl-1,4-diazepan-2-one as a novel pharmacophore for potent and selective DPP-4 inhibitors, optimization of this new lead with different substitution on the seven-membered ring resulted in several highly potent and selective, orally bioavailable, and efficacious DPP-4 inhibitors, such as 3R-methyl-1-cyclopropyl-1,4-diazepan-2-one derivative 9i (DPP-4 IC(50)=8.0 nM) and 3R,6R-dimethyl-1,4-diazepan-2-one derivative 14a (DPP-4 IC(50)=9.7 nM).     

Pyrazole-based cathepsin S inhibitors with improved cellular potency

High potency pyrazole-based noncovalent inhibitors of human cathepsin S (CatS) were developed by modification of the benzo-fused 5-membered ring heterocycles found in earlier series of CatS inhibitors. Although substitutions on this heterocyclic framework had a moderate impact on enzymatic potency, dramatic effects on cellular activity were observed. Optimization afforded indole- and benzothiophene-derived analogues that were high affinity CatS inhibitors (IC(50)=20-40 nM) with good cellular potency (IC(50)=30-340 nM).     

4,4'-Benzophenone-O,O'-disulfamate: a potent inhibitor of steroid sulfatase

We investigated whether the benzophenone moiety can be used as core element of steroid sulfatase (STS) inhibitors. While 4- and 3-benzophenone-O-sulfamates inhibit STS with IC(50) values between 5 and 7 microM irrespective of additional hydroxy and methoxy substituents at the second phenyl ring, benzophenone-O,O'-disulfamates show increased activity. With an IC(50) value of 190 nM the 4,4'-derivative is the first small monocyclic STS inhibitor coming close to the potency of the steroidal standard estrone sulfamate.     

Benzyl vinylogous amide substituted aryldihydropyridazinones and aryldimethylpyrazolones as potent and selective PDE3B inhibitors

Aryldihydropyridazinones and aryldimethylpyrazolones with 2-benzyl vinylogous amide substituents have been identified as potent PDE3B subtype selective inhibitors. Dihydropyridazinone 8a (PDE3B IC(50)=0.19 nM, 3A IC(50)=1.3 nM) was selected for in vivo evaluation of lipolysis induction, metabolic rate increase, and cardiovascular effects.     

Knowledge-based design of 7-azaindoles as selective B-Raf inhibitors

The synthesis of a 7-azaindole series of novel, potent B-Raf kinase inhibitors using knowledge-based design was carried out. Compound 6h exhibits not only excellent potency in both the enzyme assay (IC(50)=2.5 nM) and the cellular assay (IC(50)=63 nM), but also has an outstanding selectivity profile against other kinases.     

Novel macrocyclic C-aryl glucoside SGLT2 inhibitors as potential antidiabetic agents

Novel macrocyclic C-aryl glucoside SGLT2 inhibitors were designed and synthesized. Two different synthetic routes of macrocyclization were adopted to prepare novel ansa SGLT2 inhibitors. Among the compounds tested, [1,7]dioxacyclopentadecine macrocycles possessing methylthiophenyl at the distal ring 40 or ethoxyphenyl at the distal ring 23 showed the best in vitro inhibitory activity in this series to date (40, IC(50)=0.778 nM and 23, IC(50)=0.899 nM) against hSGLT2.     

Purification and properties of Arabidopsis thaliana type 1 protein phosphatase (PP1).

The Arabidopsis thaliana type 1 protein phosphatase (PP1) catalytic subunit was released from its endogenous regulatory subunits by ethanol precipitation and purified by anion exchange and microcystin affinity chromatography. The enzyme was identified by MALDI-TOF mass spectrometry from a tryptic digest of the purified protein as a mixture of PP1 isoforms (TOPP 1-6) indicating that at least 4-6 of the eight known PP1 proteins are expressed in sufficient quantities for purification from A. thaliana suspension cells. The enzyme had a final specific activity of 8950 mU/mg using glycogen phosphorylase a as substrate, had a subunit molecular mass of 35 kDa as determined by SDS-PAGE and behaved as a monomeric protein of approx. 39 kDa on Superose 12 gel filtration chromatography. Similar to the mammalian type 1 protein phosphatases, the A. thaliana enzyme was potently inhibited by Inhibitor-2 (IC(50)=0.65 nM), tautomycin (IC(50)=0.06 nM), microcystin-LR (IC(50)=0.01 nM), nodularin (IC(50)=0.035 nM), calyculin A (IC(50)=0.09 nM), okadaic acid (IC(50)=20 nM) and cantharidin (IC(50)=60 nM). The enzyme was also inhibited by fostriecin (IC(50)=22 microM), NaF (IC(50)=2.1 mM), Pi (IC(50)=9.5 mM), and PPi (IC(50)=0.07 mM). Purification of the free catalytic subunit allowed it to be used to probe protein phosphatase holoenzyme complexes that were enriched on Q-Sepharose and a microcystin-Sepharose affinity matrix and confirmed several proteins to be PP1 targeting subunits.     

Structure-activity relationships of aryloxyalkanoic acid hydroxyamides as potent inhibitors of histone deacetylase

Syntheses of aryloxyalkanoic acid hydroxyamides are described, all of which are potent inhibitors of histone deacetylase, some being more potent in vitro than trichostatin A (IC(50)=3 nM). Variation of the substituents on the benzene ring as well as fusion of a second ring have marked effects on potency, in vitro IC(50) values down to 1 nM being obtained.     

Synthesis and biological evaluation of glycogen synthase kinase 3 (GSK-3) inhibitors: an fast and atom efficient access to 1-aryl-3-benzylureas

The glycogen synthase kinase 3 (GSK-3) is implicated in multiple cellular processes and has been linked to the pathogenesis of Alzheimer's disease (AD). In the course of our research topic we synthesized a library of potent GSK-3 inhibitors. We utilized the urea scaffold present in the potent and highly selective GSK-3 inhibitor AR-A014418 (AstraZeneca). This moiety suits both (a) a convergent approach utilizing readily accessible building blocks and (b) a divergent approach based on a microwave heating assisted Suzuki coupling. We established a chromatography-free purification method to generate products with sufficient purity for the biological assays. The structure-activity relationship of the library provided the rationale for the synthesis of the benzothiazolylurea 66 (IC(50)=140 nM) and the pyridylurea 62 (IC(50)=98 nM), which displayed two to threefold enhanced activity versus the reference compound 18 (AR-A014418: IC(50)=330 nM) in our assays.     

Phosphorylation of protein phosphatase type-1 inhibitory proteins by integrin-linked kinase and cyclic nucleotide-dependent protein kinases

Protein phosphatases play key roles in cellular regulation and are subjected to control by protein inhibitors whose activity is in turn regulated by phosphorylation. Here we investigated the possible regulation of phosphorylation-dependent type-1 protein phosphatase (PP1) inhibitors, CPI-17, PHI-1, and KEPI, by various kinases. Protein kinases A (PKA) and G (PKG) phosphorylated CPI-17 at the inhibitory site (T38), but not PHI-1 (T57). Phosphorylated CPI-17 inhibited the activity of both the PP1 catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MPH) with IC(50) values of 1-8 nM. PKA predominantly phosphorylated a site distinct from the inhibitory T73 in KEPI, whereas PKG was ineffective. Integrin-linked kinase phosphorylated KEPI (T73) and this dramatically increased inhibition of PP1c (IC(50)=0.1 nM) and MPH (IC(50)=8 nM). These results suggest that the regulatory phosphorylation of CPI-17 and KEPI may involve distinct kinases and signaling pathways.     

Role of L-type calcium channels and PKC in active tone development in rabbit coronary artery

The present study investigated active tone development in isolated ring segments of rabbit epicardial coronary artery. Endothelium-denuded (E-) or endothelium-intact (E+) vessels treated with the NO synthase inhibitor N(omega)-nitro-L-arginine (100 microM) developed active tone, which was enhanced by stretch and reversed by the NO donor sodium nitroprusside (SNP; IC(50)=9 nM). Nifedipine abolished active tone and the contractile response to phorbol dibutyrate (PDBu; 10 nM) with the same potency (IC(50)=8 nM), whereas 300 nM PDBu responses were only partially blocked by nifedipine. The classical and novel PKC inhibitors GF-109203X (IC(50)=1-2 microM) and chelerythrine (IC(50)=4-5 microM) and the classical PKC inhibitor G?-6976 (IC(50)=0.3-0.4 microM) blocked both active tone and 10 nM PDBu responses with similar potency. Active tone development was associated with depolarization of membrane potential (E(m)) and a shift to the left of the E(m)-vs.-contraction relationship determined by varying extracellular potassium. The depolarization and leftward shift were reversed by either chelerythrine (10 microM) or SNP (30 nM). PDBu (100-300 nM) increased peak L-type calcium channel (Ca(v)) currents in isolated coronary myocytes, and this effect was reversed by chelerythrine (1 microM) or G?-6976 (200 nM). SNP (500 nM) reduced Ca(v) currents only in the presence of the PKA blocker 8-bromo-2'-O-monobutyryl-cAMPS, Rp isomer (10 microM). In conclusion, active tone development in coronary artery is suppressed by basal NO release and is dependent on both enhanced Ca(v) activity and classical PKC activity. Both E(m)-dependent and -independent processes contribute to contraction. Our results suggest that one E(m)-independent process is direct enhancement of Ca(v) current by PKC.     

Synthesis and evaluation of analogues of estrone-3-O-sulfamate as potent steroid sulfatase inhibitors

Estrone sulfamate (EMATE) is a potent irreversible inhibitor of steroid sulfatase (STS). In order to further expand SAR, the compound was substituted at the 2- and/or 4-positions and its 17-carbonyl group was also removed. The following general order of potency against STS in two in vitro systems is observed for the derivatives: The 4-NO(2) > 2-halogens, 2-cyano > EMATE (unsubstituted)>17-deoxyEMATE > 2-NO(2) > 4-bromo>2-(2-propenyl), 2-n-propyl > 4-(2-propenyl), 4-n-propyl > 2,4-(2-propenyl)= 2,4-di-n-propyl. There is a clear advantage in potency to place an electron-withdrawing substituent on the A-ring with halogens preferred at the 2-position, but nitro at the 4-position. Substitution with 2-propenyl or n-propyl at the 2- and/or 4-position of EMATE, and also removal of the 17-carbonyl group are detrimental to potency. Three cyclic sulfamates designed are not STS inhibitors. This further confirms that a free or N-unsubstituted sulfamate group (H(2)NSO(2)O-) is a prerequisite for potent and irreversible inhibition of STS as shown by inhibitors like EMATE and Irosustat. The most potent derivative synthesized is 4-nitroEMATE (2), whose IC(50)s in placental microsomes and MCF-7 cells are respectively 0.8 nM and 0.01 nM.