Rational design of pyridyl derivatives of vanillin for the treatment of sickle cell disease

Hypoxia-induced polymerization of sickle hemoglobin (Hb S) is the principal phenomenon that underlays the pathophysiology and morbidity associated with sickle cell disease (SCD). Opportunely, as an allosteric protein, hemoglobin (Hb) serves as a convenient and potentially critical druggable target. Consequently, molecules that prevent Hb S polymerization (Hb modifiers), and the associated erythrocyte sickling have been investigated–and retain significant interest–as a viable therapeutic strategy for SCD. This group of molecules, including aromatic aldehydes, form high oxygen affinity Schiff-base adducts with Hb S, which are resistant to polymerization. Here, we report the design and synthesis of novel potent antisickling agents (SAJ-009, SAJ-310 and SAJ-270) based on the pharmacophore of vanillin and INN-312, a previously reported pyridyl derivative of vanillin. These novel derivatives exhibited superior in vitro binding and pharmacokinetic properties compared to vanillin, which translated into significantly enhanced allosteric and antisickling properties. Crystal structure studies of liganded Hb in the R2 quaternary state in complex with SAJ-310 provided important insights into the allosteric and antisickling properties of this group of compounds. While these derivatives generally show similar in vitro biological potency, significant structure-dependent differences in their biochemical profiles would help predict the most promising candidates for successful in vivo pre-clinical translational studies and inform further structural modifications to improve on their pharmacologic properties.     

Vanillins--a novel family of DNA-PK inhibitors

Non-homologous DNA end-joining (NHEJ) is a major pathway of double strand break (DSB) repair in human cells. Here we show that vanillin (3-methoxy-4-hydroxybenzaldehyde)—a naturally occurring food component and an acknowledged antimutagen, anticlastogen and anticarcinogen—is an inhibitor of NHEJ. Vanillin blocked DNA end-joining by human cell extracts by directly inhibiting the activity of DNA-PK, a crucial NHEJ component. Inhibition was selective and vanillin had no detectable effect on other steps of the NHEJ process, on an unrelated protein kinase or on DNA mismatch repair by cell extracts. Subtoxic concentrations of vanillin did not affect the ATM/ATR-dependent phosphorylation of Chk2 or the S-phase checkpoint response after ionising radiation. They significantly potentiated the cytotoxicity of cisplatin, but did not affect sensitivity to UVC. A limited screen of structurally related compounds identified two substituted vanillin derivatives that were 100- and 50-fold more potent than vanillin as DNA-PK inhibitors. These compounds also sensitised cells to cisplatin. The inhibition of NHEJ is consistent with the antimutagenic and other biological properties of vanillin, possibly altering the balance between DSB repair by NHEJ and homologous recombination.     

Synthesis and antimicrobial evaluation of new chalcones containing piperazine or 2,5-dichlorothiophene moiety

Two new series of chalcones have been synthesized by reacting 1-(4-piperazin-1-yl-phenyl)ethanone and 1-(2,5-dichloro-3-thienyl)-1-ethanone with different substituted benzaldehydes in turn by Claisen-Schmidt condensation. The compounds have been characterized by IR, (1)H NMR spectral and microanalysis data. All the synthesized compounds have been evaluated for antimicrobial activity. Some of these derivatives are potentially active against Gram-positive bacteria, Staphylococcus aureus and Escherichia coli while the most potent compound (1) in this study showed MIC(50) value of 2.22 microg/mL against Candida albicans.     

Exploration of the P1 SAR of aldehyde cathepsin K inhibitors

The synthesis and biological activity of a series of aldehyde inhibitors of cathepsin K are reported. Exploration of the properties of the S(1) subsite with a series of alpha-amino aldehyde derivatives substituted at the P(1) position afforded compounds with cathepsin K IC(50)s between 52 microM and 15 nM.     

Synthesis,Biological Evaluation and in Silico Studies of 3-Hydroxy-N-(2-(substituted phenyl)-4-oxothiazolidin-3-yl)-2-napthamide Derivatives

In the present study, a series of 3-hydroxy-N-(2-(substituted phenyl)-4-oxothiazolidin-3-yl)-2-napthamide derivatives were synthesized, characterized and evaluated for theirin vitroactivity, i. e., antimicrobial, antioxidant and anti-inflammatory. The target compounds were synthesized by condensation reaction of 3-hydroxy-2-naphthoic acid hydrazide with substituted benzaldehydes which were subjected to cyclization reaction with thioglycolic acid and ZnCl₂ to get target compounds. The synthesized 3-hydroxy-N-(2-(substituted phenyl)-4-oxothiazolidin-3-yl)-2-napthamide derivatives were examined for their antimicrobial activity and 3-hydroxy-N-(4-oxo-2-(3,4,5-trimethoxyphenyl)thiazolidin-3-yl)-2-naphthamide ( S20 ) exhibited the highest antimicrobial potential. The N′-(2,3-dichlorobenzylidene)-3-hydroxy-2-naphthohydrazide ( S5 ) displayed good antifungal potential against Rhizopus oryzae, whereas N′-(2,3-dichlorobenzylidene)-3-hydroxy-2-naphthohydrazide ( S20 ) showed the highest antioxidant potential and N-(2-(2,6-dichlorophenyl)-4-oxothiazolidin-3-yl)-3-hydroxy-2-naphthamide ( S16 ) displayed the highest anti-inflammatory activity. The results of molecular docking studies revealed that existence of hydrogen bonding and hydrophobic interactions with their respective proteins. In silico ADMET studies were carried out by Molinspiration, Pre-ADMET and OSIRIS property explorer to predict the pharmacokinetic behaviour of synthesized 3-hydroxy-N-(2-(substituted phenyl)-4-oxothiazolidin-3-yl)-2-napthamide derivatives.     

Remarkable positional (regio)specificity of xanthine oxidase and some dehydrogenases in the reactions with substituted benzaldehydes

Upon an increase in the size of the substituent, the reactivity of xanthine oxidase to ortho-substituted benzaldehydes drastically decreases while that to para-substituted benzaldehydes does not change significantly. The enzyme exhibits this regiospecificity with respect to both electron-withdrawing substituents (e.g., halogens) and electron-donating ones (alkyls and alkoxyls). Xanthine oxidase-catalyzed oxidation of m- and p-nitrobenzaldehyde is more than 300-times faster than that of the o-isomer, whereas the rates of their non-enzymatic oxidation are comparable, as are the rates of the enzymatic oxidation of p- and o-nitrocinnamaldehyde. These and other findings of this work indicate that the discovered positional specificity of xanthine oxidase is due to steric hindrances in the reaction of the enzyme's active center with the aldehyde moiety having a bulky substituent in its close proximity. Such regiospecificity of the enzyme exists regardless of the nature of the electron acceptor used and can be employed for the separation of mixtures of positional isomers of substituted benzaldehydes. A marked positional specificity in the xanthine oxidase-catalyzed oxidation of substituted benzaldehydes appears to be a rather general phenomenon: three other enzymes tested, alcohol dehydrogenases from horse liver and yeast and aldehyde dehydrogenase from yeast, all follow a similar pattern in the reactions with para- and ortho-substituted halobenzaldehydes.     

Carbohydrazones of substituted salicylaldehydes as potential lead compounds for the development of narrow-spectrum antimicrobials

Certain substituted salicylaldehydes are known to have highly potent antimicrobial activity against bacteria and fungi, but the mechanism underlying this remarkable activity is not known, and almost nothing has been reported on the effects of further modification of the structures, such as the formation of hydrazone-type derivatives. We report now a study on the antimicrobial properties of the carbohydrazone derivatives of several substituted salicylaldehydes. The compounds studied were synthesized from ring-substituted salicylaldehydes and carbohydrazide in the mole ratio 2:1. They were tested against Aspergillus niger, Bacillus cereus, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Saccharomyces cerevisiae and Staphylococcus epidermidis using the agar diffusion method. The carbohydrazone derived from 2,3,4-trihydroxybenzaldehyde had distinctly higher activity than the parent aldehyde in the same molar concentration. This activity was limited to one test organism (S. epidermidis), while the free aldehyde had at least some (in some cases even high) activity against all of the microbes studied. All other ones of the effective carbohydrazone compounds were distinctly less active than the parent salicylaldehydes as such. The hydrazones studied had in general a narrower antimicrobial spectrum than the free aldehydes and are thus of interest as potential lead compounds for the development of narrow-spectrum anti-microbial drugs. The mechanism of action of the aldehydes as well as that of the carbohydrazones is discussed.     

Structural insight into interaction between C20 phenylalanyl derivative of tylosin and ribosomal tunnel

Macrolides are clinically important antibiotics that inhibit protein biosynthesis on ribosomes by binding to ribosomal tunnel. Tylosin belongs to the group of 16-membered macrolides. It is a potent inhibitor of translation whose activity is largely due to reversible covalent binding of its aldehyde group with the base of A2062 in 23S ribosomal RNA. It is known that the conversion of the aldehyde group of tylosin to methyl or carbinol groups dramatically reduces its inhibitory activity. However, earlier we obtained several derivatives of tylosin having comparable activity in spite of the fact that the aldehyde group of tylosin in these compounds was substituted with an amino acid or a peptide residue. Details of the interaction of these compounds with the ribosome that underlies their high inhibitory activity were not known. In the present work, the structure of the complex of tylosin derivative containing in position 20 the residue of ethyl ester of 2-imino(oxy)acetylphenylalanine with the tunnel of the E. coli ribosome was identified by means of molecular dynamics simulations, which could explain high biological activity of this compound.     

Synthesis and evaluation of novel 4-[(3H,3aH,6aH)-3-phenyl)-4,6-dioxo-2-phenyldihydro-2H-pyrrolo[3,4-d]isoxazol-5(3H,6H,6aH)-yl]benzoic acid derivatives as potent acetylcholinesterase inhibitors and anti-amnestic agents

The present study was designed to synthesize and evaluate pyrrolo-isoxazole benzoic acid derivatives as potential acetylcholinesterase (AChE) inhibitors for the management of Alzheimer's disease. The synthesis of pyrrolo-isoxazole benzoic acid derivatives involved ring opening cyclization of p-aminobenzoic acid with maleic anhydride to yield maleanilic acid, which in turn afforded N-arylmaleimide via ring closed cyclization. Azomethine-N-oxides were obtained by condensation of N-arylhydroxylamine with differently substituted benzaldehydes followed by refluxing of N-arylmaleimide with differently substituted azomethine-N-oxides to pyrrolo-isoxazole benzoic acid derivatives as cis- and trans-stereoisomers. The synthesized compounds were evaluated in vitro for AChE inhibitory activity in rat brain homogenate with donepezil as standard AChE inhibitor. Thereafter, the most potent test compound was evaluated for in vitro butyrylcholinesterase inhibitory activity and in vivo memory evaluation in scopolamine (0.4mg/kg)-induced amnesia in mice by employing Morris water maze test. All pyrrolo-isoxazole benzoic acid derivatives demonstrated potent AChE inhibitory activity. Most of compounds exhibited similar activity to donepezil and four of them (7h, 7i, 8i, and 8h, IC(50)=19.1±1.9-17.5±1.5nM) displayed higher inhibitory activity as compared to donepezil (21.5±3.2nM) with compound 8ia (IC(50)=17.5±1.5nM) being the most active one. The test compound 8ia also ameliorated scopolamine-induced amnesia in mice in terms of restoration of time spent in target quadrant (TSTQ) and escape latency time (ELT). It may be concluded that pyrrolo-isoxazole benzoic acid derivatives may be employed as potential AChE inhibitors.     

Inhibitory effects of flavonoids on aldehyde oxidase activity

Flavonoids are an important group of natural compounds that can interfere with the activity of some enzymes. In this study, effects of various flavonoids on aldehyde oxidase (AO) activity were evaluated in vitro. AO was partially purified from guinea pig liver. The effects of 12 flavonoids from three subclasses of flavon-3-ol, flavan-3-ol and flavanone on the oxidation of vanillin and phenanthridine as substrates of AO and xanthine as a substrate of xanthine oxidase (XO) were investigated spectrophotometrically. Among the 12 flavonoids, myricetin and quercetin were the most potent inhibitors of both AO and XO. In general, the oxidation of vanillin was more inhibited by flavonoids than that of phenanthridine. Almost all of the flavonoids inhibited AO activity more potently than XO, which was more evident with non-planner flavanols. A planner structure seems to be essential for a potent inhibitory effect and any substitution by sugar moieties reduces the inhibitory effects. This study could provide a new insight into AO natural inhibitors with potential to lead to some food-drug interactions.     

Inhibitory effects of flavonoids on aldehyde oxidase activity

Flavonoids are an important group of natural compounds that can interfere with the activity of some enzymes. In this study, effects of various flavonoids on aldehyde oxidase (AO) activity were evaluated in vitro. AO was partially purified from guinea pig liver. The effects of 12 flavonoids from three subclasses of flavon-3-ol, flavan-3-ol and flavanone on the oxidation of vanillin and phenanthridine as substrates of AO and xanthine as a substrate of xanthine oxidase (XO) were investigated spectrophotometrically. Among the 12 flavonoids, myricetin and quercetin were the most potent inhibitors of both AO and XO. In general, the oxidation of vanillin was more inhibited by flavonoids than that of phenanthridine. Almost all of the flavonoids inhibited AO activity more potently than XO, which was more evident with non-planner flavanols. A planner structure seems to be essential for a potent inhibitory effect and any substitution by sugar moieties reduces the inhibitory effects. This study could provide a new insight into AO natural inhibitors with potential to lead to some food-drug interactions.     

Transition-state analogues as inhibitors of L-dopa decarboxylase

1. A series of compounds has been prepared which are analogues of the transition state of the reaction catalysed by L-dopa decarboxylase (EC 4.1.1.28). 2. These compounds are reduced adducts of the substrate (L-dopa) and coenzyme (pyridoxal phosphate), as well as analogues of these substances (D-dopa, pyridoxal and salicaldehyde). 3. Compounds were also prepared with an oxazine link between the 3'-oxygen and the nitrogen attached to the 4'-carbon of the aldehyde moiety. 4. None of the D-dopa adducts produced any significant inhibition, but the L-dopa adducts were all active at millimolar levels, with the oxazine derivatives being more active than their parent compounds. 5. Inhibition was competitive with respect to L-dopa, but was neither competitive nor non-competitive with respect to pyridoxal phosphate. 6. The most active compound tested was the oxazine derivative of the L-dopa/salicaldehyde adduct, with an estimated Ki of 58.0 microM. 7. Increased inhibitory activity was observed when enzyme depleted of pyridoxal phosphate was used.     

Substrate range of acetohydroxy acid synthase I from Escherichia coli in the stereoselective synthesis of alpha-hydroxy ketones

Acetohydroxy acid synthase I appears to be the most effective of the AHAS isozymes found in Escherichia coli in the chiral synthesis of phenylacetyl carbinol from pyruvate and benzaldehyde. We report here the exploration of a range of aldehydes as substrates for AHAS I and demonstrate that the enzyme can accept a wide variety of substituted benzaldehydes, as well as heterocyclic and heteroatomic aromatic aldehydes, to produce chiral carbinols. The active site of AHAS I does not appear to impose serious steric constraints on the acceptor substrate. The influence of electronic effects on the reaction has been probed using substituted benzaldehydes as substrates. The electrophilicity of the aldehyde acceptor substrates is most important to their reactivity, but the lipophilicity of substituents also affects their reactivity. AHAS I is an effective biosynthetic platform for production of a variety of alpha-hydroxy ketones, compounds with considerable potential as pharmacological precursors.     

Antiviral activity of pyridyl imidazolidinones against enterovirus 71 variants

Pyridyl imidazolidinone is a novel class of capsid binder which can inhibit enterovirus 71 (EV71). In this study, we tested the susceptibility of six recombinant viruses with different single-site mutations in VP1. Eleven modified pyridyl imidazolidinones were synthesized and used to probe the interaction between these compounds and the EV71 VP1 protein. We found that the D31N or E98K mutant viruses were susceptible to bulkier compounds, which suggested that mutations at these two sites in VP1 may widen the hydrophobic pocket of VP1, allowing bulkier compounds to enter and interfere VP1-receptor binding. Additionally, the Y116H mutant was more resistant to pyridyl imidazolidinone compounds containing a methyl group in the central position of the hydrophobic linker. When a trifluoromethyl group was substituted for the methyl group in the middle of the linker chain, the inhibitory effect was totally abolished in the Y116H mutant, suggesting that the interaction between Tyr (Y) 116 of VP1 and the central position of the linker chain of pyridyl imidazolodinone is very important for drug efficacy. A V192M mutant was resistant to most of the derivatives, indicating that residue 192 is a key mutation for resistance to pyridyl imidazolidinone.     

Orally active CCR5 antagonists as anti-HIV-1 agents. Part 3: Synthesis and biological activities of 1-benzazepine derivatives containing a sulfoxide moiety

In order to develop orally active CCR5 antagonists, 1-propyl- or 1-isobutyl-1-benzazepine derivatives containing a sulfoxide moiety have been designed, synthesized, and evaluated for their biological activities. Sulfoxide compounds containing a 2-pyridyl group were first investigated, which led to discovering that the presence of a methylene group between the sulfoxide moiety and 2-pyridyl group was necessary for increased inhibitory activity in a binding assay. After further chemical modification, it was found that replacement of the pyridyl group with an imidazolyl or 1,2,4-triazolyl group enhanced activity in the binding assay and that S-sulfoxide compounds were more active than R-isomers. Particularly, compounds (S)-4r, (S)-4s, and (S)-4w exhibited highly potent CCR5 antagonistic activities (IC50=1.9, 1.7, 1.6 nM, respectively) and inhibitory effects (IC50=1.0, 2.8, 7.7 nM, respectively) in the HIV-1 envelope mediated membrane fusion assay, together with good pharmacokinetic properties in rats. In addition, we established the synthesis of (S)-4r and (S)-4w by asymmetric oxidation with titanium-(S)-(-)-1,1'-bi-2-naphthol complex.     

Synthesis and pharmacological evaluation of coumarin derivatives as cannabinoid receptor antagonists and inverse agonists

In the present study we synthesized 36 coumarin and 2H-chromene derivatives applying a recently developed umpoled domino reaction using substituted salicylaldehyde and α,β-unsaturated aldehyde derivatives as starting compounds. In radioligand binding studies 5-substituted 3-benzylcoumarin derivatives showed affinity to cannabinoid CB₁ and CB₂ receptors and were identified as new lead structures. In further GTPγS binding studies selected compounds were shown to be antagonists or inverse agonists.     

Structure-based development of pyridoxal propionate derivatives as specific inhibitors of cathepsin K in vitro and in vivo

We found that pyridoxal phosphate shows considerable inhibition of cathepsins. CLIK-071, in which the phosphate ester of position 3 of pyridoxal phosphate was replaced by propionate, strongly inhibited cathepsin B. Three new types of synthetic pyridoxal propionate derivatives showing specific inhibition of cathepsin K were developed. New synthetic pyridoxal propionate derivatives, -162, -163, and -164, in which the methyl arm of position 6 of CLIK-071 was additionally modified, strongly inhibited cathepsin K and cathepsin S weakly, but other cathepsins were not inhibited. CLIK-166, in which the position 4 aldehyde of CLIK-071 is replaced by a vinyl radical and position 5 is additionally modified, showed cathepsin K-specific inhibition at 10(-5) M. Pit formation due to bone collagen degradation by cathepsin K of rat osteoclasts was specifically suppressed by administration of CLIK-164, but not by inhibitors of cathepsin L or B.     

Preliminary evaluation of the cytotoxicity of a series of tris-2-aminoethylamine (Tren) based hexadentate heterocyclic donor agents

Tachpyridine is a cytotoxic metal chelator with potential anti-tumor activity. The synthesis and evaluation of a set of derivatives of the related hexadentate heterocyclic donor agents tris-2-aminoethylamine (tren) and tris[N-(2-pyridylmethylene)-2-aminoethyl]amine (trenpyr) was performed to compare their cytotoxic activity to tachpyridine in HeLa tumor cells. Methyl groups were added to the pyridyl ring of trenpyr, and the effects of alkyl group substitution on cell survival were assessed. Profound cytotoxicity was observed and IC50 data were obtained in ascending order from those compounds substituted with a methyl group at the 3-, 4-, or 5-position and lastly by the 6-methyl derivative. These results suggest that analogous derivatives with substitution at the 3-position of the pyridyl ring deserve further exploration.     

Biotransformation of protocatechuic aldehyde and caffeic acid to vanillin and capsaicin in freely suspended and immobilized cell cultures of Capsicum frutescens

Freely suspended cells and immobilized cell cultures of Capsicum frutescens Mill. were treated with phenylpropanoid intermediates--protocatechuic aldehyde and caffeic acid to study their biotransformation ability. It was found that externally fed protocatechuic aldehyde and caffeic acids were biotransformed to vanillin and capsaicin. It was noted that this culture biotransformed externally fed protocatechuic aldehyde to vanillin more than its conversion to capsaicin, whereas, caffeic acid-treated cultures accumulated more capsaicin than vanillin. The maximum accumulation of vanillin (5.63 mg l(-1)) and capsaicin (3.83 mg l(-1)) was recorded on the 6th and 15th day, respectively in immobilized C. frutescens cell cultures treated with protocatechuic aldehyde, which was 1.8 and 1.4 times higher than in protocatechuic aldehyde-treated freely suspended cell cultures. Caffeic acid-treated immobilized C. frutescens cell cultures accumulated maximum vanillin and capsaicin at 2.68 and 3.03 mg l(-1) culture, respectively, on the 9th and 12th day, which was 1.65 and 1.33 times over freely suspended cultures treated with caffeic acid. The addition of S-adenosyl-L-methionine, a methyl donor, to protocatechuic aldehyde-treated immobilized C. frutescens cell cultures, resulted in accumulation of vanillin (14.08 mg l(-1)) on the 4th day, which was 2.5-fold higher than that in cultures treated with protocatechuic aldehyde alone, suggesting the influence of S-adenosyl-L-methionine on O-methylation of protocatechuic aldehyde, resulting in more vanillin accumulation. The increase in vanillin accumulation was well correlated with an increase in specific activity of caffeic acid O-methyltransferase in protocatechuic aldehyde and S-adenosyl-L-methionine-treated immobilized C. frutescens cell cultures. This study also provides an example for an alternative route to formation of vanillin by C. frutescens cell cultures.