A facile and chemoselectivity in synthesis of 4-chloro-N-(4-((1-hydroxy-2-methylpropan-2-yl)oxy)phenethyl)benzamide, the alcohol derivative of Bezafibrate.

A facile method for the reduction of carboxylic acid group of Bezafibrate, an approved drug, is described. The selective reduction of carboxylic acid group to corresponding alcohol was carried out by activation of the carboxylic acid moiety via mixed anhydride followed by the addition of stoichiometric amount of NaBH4 and methanol to obtain the first alcohol variant of Bezafibrate. The reaction was completed in 5-10 min in excellent yield and purity. The new alcohol derivative was characterized by spectroscopic methods. This is the first report on this new molecule.

Chemoselective Reduction of Fenofibric Acid to Alcohol in the Presence of Ketone by Mixed Anhydride and Sodium Borohydride.

A highly efficient and facile protocol for the selective reduction of carboxylic acid of Fenofibric acid to corresponding alcohol was developed. The selective reduction was carried out by activation of carboxylic acid by mixed anhydride followed by the reaction of sodium borohydride in presence of methanol. This is the first example of chemoselective reduction of carboxylic acid to alcohol in presence of a ketone without any external catalyst or ligand in a single step. The reaction offers wide applicability for the selective carboxylic group reduction methodology. The chemoselective reduction was demonstrated by the reduction of Fenofibric acid, an active metabolite of the drug Fenofibrate, to corresponding alcohol in excellent selectivity, yield, and purity.

Signature of Glycylglutamic Acid Structure.

BACKGROUND: Glutamate (Glu) is of great interest in biomedical research. It is considered a biomarker in diabetes, which may potentially contribute to the development of autism in genetically vulnerable human populations, and it is found in relation to advanced glycation end products (AGEs) [1]. Additionally, Glu plays an active role in the function of ligand-gated ion channel glutamate receptors, chloride channels capable of filtering glutamate, as well as Potassium (K+)-channel [2]. Glu attains α [3] and ß [4] crystal forms and Cß-CH2 show asymmetric 1H signal pattern in NMR spectra. OBJECTIVES: The current study was undertaken to understand the signal patterns of Cß-CH2 in Glu of the smallest dipeptide, Glycylglutamic Acid (GlyGlu), as well as the order, and planarity of the amide bond in the molecule. MATERIALS AND METHODS: NMR spectra of GlyGlu were measured in D2O to deduce 1H and 13C chemical shifts and coupling constants. GlyGlu was crystallized from MeOH and the structure was determined by single crystal X-ray diffraction techniques. RESULTS: The sidechain of Glu in the dipeptide dissimilates the ß form. The amino group of Gly (Glycine) is protonated and exhibits hydrogen bonding with the main chain carboxylate group of a symmetry-related Glu that is deprotonated in the crystal packing of GlyGlu. The deprotonated main chain carboxylate of Glu is also in hydrogen-bonding distance from the side chain carboxylic acid group that is in the protonated form of a symmetry-related Glu of the dipeptide. The Cß-CH2 geminal protons on the side chain of Glu have different chemical shifts and splitting pattern in 1H NMR reflecting their dissymmetric environment. CONCLUSION: The results reported will be useful for monitoring changes that Glu and/or molecules in connection to Glu may undergo in in vivo, in situ, and in vitro conditions. This provides a valuable metric which will enable the examination of the metabolites relevant to the detection and diagnosis of disease or developmental conditions, as well as scrutinizing the effectiveness of treatment options.

Synthesis and Chiral Separation of Fibratol, Isopropyl 2-(4-((4-chlorophenyl)(hydroxyl) methyl)-phenoxy)-2-methylpropanoate.

Practical synthetic route for the formation of enantiomeric mixture of Isopropyl 2-(4-((4-chlorophenyl)(hydroxyl)methyl)phenoxy)-2-methylpropanoate (Fibratol 2a/b) from isopropyl 2-(4-(4-chlorobenzoyl)phenoxy)-2-methylpropanoate (Fenofibrate 1) has been developed. Method has also been established for the chiral separation of enantiomers of Fibratol 2a/b that is synthesized using the route mentioned above. The optical activity determined for enantiomerically separated Fibratol (2a) and Fibratol (2b) are -5.2° and 8.0° which reflect their ability to rotate plane polarized light counterclockwise (levo) and clockwise (dextro), respectively.

Characterization of WY 14,643 and its Complex with Aldose Reductase.

The peroxisome proliferator, WY 14,643 exhibits a pure non-competitive inhibition pattern in the aldehyde reduction and in alcohol oxidation activities of human Aldose reductase (hAR). Fluorescence emission measurements of the equilibrium dissociation constants, Kd, of oxidized (hAR•NADP+) and reduced (hAR•NADPH) holoenzyme complexes display a 2-fold difference between them. Kd values for the dissociation of WY 14,643 from the oxidized (hAR•NADP+•WY 14,643) and reduced (hAR•NADPH•WY 14,643) ternary complexes are comparable to each other. The ternary complex structure of hAR•NADP+•WY 14,643 reveals the first structural evidence of a fibrate class drug binding to hAR. These observations demonstrate how fibrate molecules such as WY 14,643, besides being valued as agonists for PPAR, also inhibit hAR.

B-factor Analysis and Conformational Rearrangement of Aldose Reductase.

The NADPH-dependent reduction of glucose reaction that is catalyzed by Aldose Reductase (AR) follows a sequential ordered kinetic mechanism in which the co-factor NADPH binds to the enzyme prior to the aldehyde substrate. The kinetic/structural experiments have found a conformational change involving a hinge-like movement of a surface loop (residues 213-224) which is anticipated to take place upon the binding of the diphosphate moiety of NADPH. The reorientation of this loop, expected to permit the release of NADP+, represents the rate-limiting step of the catalytic mechanism. This study reveals: 1) The Translation/Libration/Screw (TLS) analysis of absolute B-factors of apo AR crystal structures indicates that the 212-224 loop might move as a rigid group. 2) Residues that make the flexible loop slide in the AR binary and ternary complexes. 3) The normalized B-factors separate this segment into three different clusters with fewer residues.

Biomolecular chemistry of isopropyl fibrates.

Isopropyl 2-[4-(4-chlorobenzoyl)-phenoxy]-2-methylpropanoic acid and isopropyl 2-(4-chlorophenoxy)-2-methylpropanoate, also known as fenofibrate and isopropyl (iPr) clofibrate, are hypolipidemic agents of the fibrate family. In a previously reported triclinic structure of fenofibrate (polymorph I), the methyl groups of the iPr moiety are located symmetrically about the carboxylate group. We report a new monoclinic form (polymorph II) of fenofibrate and a first structural description of iPr clofibrate, and in these the methyl groups are placed asymmetrically about the carboxylate group. In particular, the dihedral (torsion) angle between the hydrogen atom on the secondary C and the C atom of the carboxyl group makes a 2.74° angle about the ester O···C bond in the symmetric fenofibrate structure of polymorph I, whereas the same dihedral angle is 45.94° in polymorph II and -30.9° in the crystal structure of iPr clofibrate. Gas-phase density functional theory (DFT) geometry minimizations of fenofibrate and iPr clofibrate result in lowest energy conformations for both molecules with a value of about ±30° for this same angle between the OC-O-C plane and the C-H bond of the iPr group. A survey of crystal structures containing an iPr ester group reveals that the asymmetric conformation is predominant. Although the hydrogen atom on the secondary C atom of the iPr group is located at a comparable distance from the carbonyl oxygen in the symmetric and asymmetric fenofibrate (2.52 and 2.28 Å) and the iPr clofibrate (2.36 Å) structures, this hydrogen atom participates in a puckered five-membered ring arrangement in the latter two that is unlike the planar arrangement found in symmetric fenofibrate (polymorph I). Polar molecular surface area values indicate fenofibrate and iPr clofibrate are less able to act as acceptors of hydrogen bonds than their corresponding acid derivatives. Surface area calculations show that dynamic polar molecular surface area values of the iPr esters of the fibrates are lower than those of their acids, implying that the fibrates have better membrane permeability and a higher absorbability and hence are better prodrugs when these agents need to be orally administered.

The role of Cys-298 in aldose reductase function.

Diabetic tissues are enriched in an "activated" form of human aldose reductase (hAR), a NADPH-dependent oxidoreductase involved in sugar metabolism. Activated hAR has reduced sensitivity to potential anti-diabetes drugs. The C298S mutant of hAR reproduces many characteristics of activated hAR, although it differs from wild-type hAR only by the replacement of a single sulfur atom with oxygen. Isothermal titration calorimetry measurements revealed that the binding constant of NADPH to the C298S mutant is decreased by a factor of two, whereas that of NADP(+) remains the same. Similarly, the heat capacity change for the binding of NADPH to the C298S mutant is twice increased; however, there is almost no difference in the heat capacity change for binding of the NADP(+) to the C298S. X-ray crystal structures of wild-type and C298S hAR reveal that the side chain of residue 298 forms a gate to the nicotinamide pocket and is more flexible for cysteine compared with serine. Unlike Cys-298, Ser-298 forms a hydrogen bond with Tyr-209 across the nicotinamide ring, which inhibits movements of the nicotinamide. We hypothesize that the increased polarity of the oxidized nicotinamide weakens the hydrogen bond potentially formed by Ser-298, thus, accounting for the relatively smaller effect of the mutation on NADP(+) binding. The effects of the mutant on catalytic rate constants and binding constants for various substrates are the same as for activated hAR. It is, thus, further substantiated that activated hAR arises from oxidative modification of Cys-298, a residue near the nicotinamide binding pocket.

Fibrates in the chemical action of daunorubicin.

Anthracyclines are an important reagent in many chemotherapy regimes for treating a wide range of tumors. One of the primary mechanisms of anthracycline action involves DNA damage caused by inhibition of topoisomerase II. Enzymatic detoxification of anthracycline is a major critical factor that determines anthracycline resistance. Natural product, daunorubicin a toxic analogue of anthracycline is reduced to less toxic daunorubicinol by the AKR1B10, enzyme, which is overexpressed in most cases of smoking associate squamous cell carcinoma (SCC) and adenocarcinoma. In addition, AKR1B10 was discovered as an enzyme overexpressed in human liver, cervical and endometrial cancer cases in samples from uterine cancer patients. Also, the expression of AKR1B10 was associated with tumor recurrence after surgery and keratinization of squamous cell carcinoma in cervical cancer and estimated to have the potential as a tumor intervention target colorectal cancer cells (HCT-8) and diagnostic marker for non-small-cell lung cancer. This article presents the mechanism of daunorubicin action and a method to improve the effectiveness of daunorubicin by modulating the activity of AKR1B10.

Cancer biomarker AKR1B10 and carbonyl metabolism.

A member of the aldo-keto reductase (AKR) protein superfamily, AKR1B10, is overexpressed in human liver cancers as well as in many adenocarcinoma cases due to smoking. AKR1B10 is also detected in instances of cervical and endometrial cancer in uterine cancer patients. In addition, AKR1B10 has been identified as a biomarker for non-small-cell lung cancer by a combined bioinformatics and clinical analysis. Furthermore, in breast cancer cells, fatty acid biosynthesis is regulated by AKR1B10. AKR1B10 contains 316 residues, shares 70% sequence identity with aldose reductase (AKR1B1) and has the conserved Cys residue at position 299. Carbonyl groups in some anticancer drugs and dl-glyceraldehyde are converted by AKR1B10 to their corresponding alcohols. The anticancer drug daunorubicin, which is currently used in the clinical treatment of various forms of cancer, is converted by AKR1B10 to daunorubicinol with a K(m) and k(cat) of 1.1+/-0.18 mM and 1.4+/-0.16 min(-1), respectively. This carbonyl reducing activity of AKR1B10 decreases the anticancer effectiveness of daunorubicin. Similarly, kinetic parameters K(m) and k(cat) (NADPH, DL-glyceraldehyde) for the reduction of dl-glyceraldehyde by wild-type AKR1B10 are 2.2+/-0.2 mM and 0.71+/-0.05 sec(-1), respectively. Mutation of residue 299 from Cys to Ser in AKR1B10 reduces the protein affinity for dl-glyceraldehyde and enhances AKR1B10's catalytic activity but overall catalytic efficiency is reduced. For dl-glyceraldehyde reduction that is catalyzed by the Cys299Ser mutant AKR1B10, K(m) is 15.8+/-1.0mM and k(cat) (NADPH, DL-glyceraldehyde) is 2.8+/-0.2 sec(-1). This implies that the substrate specificity of AKR1B10 is drastically affected by mutation of residue 299 from Cys to Ser. In the present paper, we use this mutation in AKR1B10 to characterize a library of compounds regarding their different inhibitory potency on the carbonyl reducing activity of wild-type and the Cys299Ser mutant AKR1B10.

Inhibiting wild-type and C299S mutant AKR1B10; a homologue of aldose reductase upregulated in cancers.

AKR1B10 is an aldose reductase (AR) homologue overexpressed in liver cancer and various forms of that enzyme in carcinomas catalyze the reduction of anticancer drugs, potential cytostatic drug, and dl-glyceraldehyde but do not catalyze the reduction of glucose. Kinetic parameters for wild-type and C299S mutant AKR1B10 indicate that substitution of serine for cysteine at position 299 reduces the affinity of this protein for dl-glyceraldehyde and enhances its catalytic activity. Fibrates suppress peroxisome proliferation and the development of liver cancer in human. Here we report the potency of fibrate-mediated inhibition of the carbonyl reduction catalyzed by wild-type and C299S mutant AKR1B10 and compare it with known AR inhibitors. Wild-type AKR1B10-catalyzed carbonyl reduction follows pure non-competitive inhibition kinetics using zopolrestat, EBPC or sorbinil, whereas fenofibrate, Wy 14,643, ciprofibrate and fenofibric acid follow mixed non-competitive inhibition kinetics. In contrast, catalysis of reaction by the C299S AKR1B10 mutant is not inhibited by sorbinil and EBPC. Despite these differences, the C299S AKR1B10 mutant still manifests kinetics similar to the wild-type protein with other fibrates including zopolrestat, fenofibrate, Wy 14,346, gemfibrozil and ciprofibrate that show mixed non-competitive inhibition kinetics. The reaction of the mutant AKR1B10 is inhibited by fenofibric acid, but manifests pure non-competitive inhibition kinetics that are different from those demonstrated for the wild-type enzyme.

Chemistory of Fibrates.

Since the description of the synthetic chemical clofibrate in 1962, various derivatives of fibrates with a diversity of chemical structures have been developed. Several of these are used clinically to treat dyslipidemia because they are generally effective in lowering elevated plasma triglycerides and cholesterol. Studies suggest that several biochemical mechanisms underlie fibrate-mediated modulation of lipoprotein and related metabolites. These mechanisms are: 1) induced lipoprotein lipolysis; 2) induced hepatic fatty acid uptake and reduced hepatic triglyceride formation; 3) amplified removal of low density lipoprotein (LDL) particles; 4) reduced neutral lipid (cholesteryl ester and triglyceride) exchange between very low density lipoprotein (VLDL) and high density lipoprotein (HDL) resulting from decreased plasma levels of triglyceride-rich lipoprotein (TRL); and 5) increased HDL production and stimulation of reverse cholesterol transport. Recent studies of structure-based inhibitor design strategy revealed that an independent enzyme, aldose reductase (AR), is a target of fibrate activity, an additional biochemical mechanism. AR has been implicated as a major player in the development of diabetes and diabetic complications because of its ability to catalyze the conversion of glucose to sorbitol. This article discusses various targets of fibrate action, biochemical pathways and commonalities in potential molecular interactions.

WY 14,643 inhibits human aldose reductase activity.

Aldose reductase (AR) is implicated to play a critical role in diabetes and cardiovascular complications because of the reaction it catalyzes. Our data reveal that peroxisome proliferator WY 14,643, follows a pure non-competitive inhibition pattern in the aldehyde reduction activity as well as in the alcohol oxidation activity of AR. This finding communicates for the first time a novel feature of WY 14,643 in regulating AR activity. In addition, this observation indicates that AR, AR-like proteins and aldo-keto reductase (AKR) members may be involved in the WY 14,643 mechanism of action when it is administered as PPAR agonist.

Crystal structure of the restriction-modification system control element C.Bcll and mapping of its binding site.

Protection from DNA invasion is afforded by restriction-modification systems in many bacteria. The efficiency of protection depends crucially on the relative expression levels of restriction versus methytransferase genes. This regulation is provided by a controller protein, named C protein. Studies of the Bcll system in E. coli suggest that C.Bcll functions as a negative regulator for M.Bcll expression, implying that it plays a role in defense against foreign DNA during virus infection. C.Bcll binds (Kd = 14.3 nM) to a 2-fold symmetric C box DNA sequence that overlaps with the putative -35 promoter region upstream of the bcllM and bcllC genes. The C.Bcll fold comprises five alpha helices: two helices form a helix-turn-helix motif, and the remaining three helices form the extensive dimer interface. The C.Bcll-DNA model proposed suggests that DNA bending might play an important role in gene regulation, and that Glu27 and Asp31 in C.Bcll might function critically in the regulation.

Fibrates inhibit aldose reductase activity in the forward and reverse reactions.

Fibrates such as bezafibrate, gemfibrozil, clofibric acid, ciprofibrate and fenofibrate, are ligands for peroxisome proliferator-activated receptor alpha (PPARalpha), and are used as therapeutic agents in the treatment of hyperlipidemia. Synthesis and accumulation of sorbitol in cells due to aldose reductase (AR) activity is implicated in secondary diabetic complications. In pursuit of finding a lead compound identification to design an effective AR inhibitor employing fragment-based design-like approach, we found that this class of compounds and their nearest neighbors could inhibit AR. Bezafibrate and gemfibrozil displayed a mixed non-competitive inhibition pattern in the glyceraldehyde reduction activity and pure non-competitive inhibition pattern in the benzyl alcohol oxidation activity of AR. Clofibric acid, ciprofibrate and fenofibrate showed pure non-competitive inhibition patterns in the forward reaction. In the reverse reaction, clofibric acid displayed a non-competitive inhibition pattern while ciprofibrate and fenofibrate displayed competitive inhibition patterns. This finding reveals for the first time a novel attribute of the fibrates in the regulation of AR activity and may be useful as lead compounds to control the function of AR in the progression and treatment of secondary diabetic complications in addition to other clinical conditions. Alternatively, these findings demonstrate that AR plays a significant role in the fibrate metabolism under various scenarios.

High-resolution crystal structure of AKR11C1 from Bacillus halodurans: an NADPH-dependent 4-hydroxy-2,3-trans-nonenal reductase.

Aldo-keto reductase AKR11C1 from Bacillus halodurans, a new member of aldo-keto reductase (AKR) family 11, has been characterized structurally and biochemically. The structures of the apo and NADPH bound form of AKR11C1 have been solved to 1.25 A and 1.3 A resolution, respectively. AKR11C1 possesses a novel non-aromatic stacking interaction of an arginine residue with the cofactor, which may favor release of the oxidized cofactor. Our biochemical studies have revealed an NADPH-dependent activity of AKR11C1 with 4-hydroxy-2,3-trans-nonenal (HNE). HNE is a cytotoxic lipid peroxidation product, and detoxification in alkaliphilic bacteria, such as B.halodurans, plays a crucial role in survival. AKR11C1 could thus be part of the detoxification system, which ensures the well being of the microorganism. The very poor activity of AKR11C1 on standard, small substrates such as benzaldehyde or DL-glyeraldehyde is consistent with the observed, very open active site lacking a binding pocket for these substrates. In contrast, modeling of HNE with its aldehyde function suitably positioned in the active site suggests that its elongated hydrophobic tail occupies a groove defined by hydrophobic side-chains. Multiple sequence alignment of AKR11C1 with the highly homologous iolS and YqkF proteins shows a high level of conservation in this putative substrate-binding site. We suggest that AKR11C1 is the first structurally characterized member of a new class of AKRs with specificity for substrates with long aliphatic tails.

Fenofibric acid.

Unlike the related fenofibrate molecule [Henry, Zhang, Gao & Bruckner (2003). Acta Cryst. E59, o699-o700], fenofibric acid {systematic name: 2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoic acid}, C17H15ClO4, contains a carboxylic acid moiety instead of an ester moiety. This polar moiety plays an important role in the formation of a rare acid-to-ketone hydrogen-bond-type packing interaction. The lack of an isopropyl group in fenofibric acid aligns the carboxyl group on the same side as the ketone carbonyl group; this conformation may play an important role in discrimination between the acid and the fenofibrate molecule in molecular recognition.

The role of glutathione in cancer.

Glutathione is an abundant natural tripeptide found within almost all cells. Glutathione is highly reactive and is often found conjugated to other molecules via its sulfhydryl moiety. It instils several vital roles within a cell including antioxidation, maintenance of the redox state, modulation of the immune response and detoxification of xenobiotics. With respect to cancer, glutathione metabolism is able to play both protective and pathogenic roles. It is crucial in the removal and detoxification of carcinogens, and alterations in this pathway, can have a profound effect on cell survival. However, by conferring resistance to a number of chemotherapeutic drugs, elevated levels of glutathione in tumour cells are able to protect such cells in bone marrow, breast, colon, larynx and lung cancers. Here we present a number of studies investigating the role of glutathione in promoting cancer, impeding chemotherapy, and the use of glutathione modulation to enhance anti-neoplastic therapy.