Design,synthesis and biological evaluation of novel inosine 5′-monophosphate dehydrogenase (IMPDH) inhibitors

Abstract

This study is based on our attempts to further explore the structure–activity relationship (SAR) of VX-148 (3) in an attempt to identify inosine 5′-mono-phosphate dehydrogenase (IMPDH) inhibitors superior to mycophenolic acid. A five-point pharmacophore developed using structurally diverse, known IMPDH inhibitors guided further design of novel analogs of 3. Several conventional as well as novel medicinal chemistry strategies were tried. The combined structure- and ligand-based approaches culminated in a few analogs with either retained or slightly higher potency. The compounds which retained the potency were also checked for their ability to inhibit human peripheral blood mononuclear cells proliferation. This study illuminates the stringent structural requirements and strict SAR for IMPDH II inhibition.     

Synthesis of the inosine 5′-monophosphate dehydrogenase (IMPDH) inhibitors

Abstract

Inosine 5′-monophosphate dehydrogenase (IMPDH) is important molecular target for potential anticancer, antiviral, antibacterial and immunosuppressive agents. A lot of compounds were obtained to establish their activity toward this enzyme, and to improve therapeutic properties of IMPDH inhibitors used as the drugs. Some of the recently reported analogs exhibited promising results during in vitro and in vivo examinations in comparison to substances applied in clinic. In this review, we describe synthesis and biological activity evaluations of the newly designed IMPDH inhibitors.     

Hit discovery of Mycobacterium tuberculosis inosine 5′-monophosphate dehydrogenase,GuaB2, inhibitors

Tuberculosis remains a global concern. There is an urgent need of newer antitubercular drugs due to the development of resistant forms of Mycobacterium tuberculosis (Mtb). Inosine 5′-monophosphate dehydrogenase (IMPDH), guaB2, of Mtb, required for guanine nucleotide biosynthesis, is an attractive target for drug development. In this study, we screened a focused library of 73 drug-like molecules with desirable calculated/predicted physicochemical properties, for growth inhibitory activity against drug-sensitive MtbH37Rv. The eight hits and mycophenolic acid, a prototype IMPDH inhibitor, were further evaluated for activity on purified Mtb-GuaB2 enzyme, target selectivity using a conditional knockdown mutant of guaB2 in Mtb, followed by cross-resistance to IMPDH inhibitor-resistant SRMV2.6 strain of Mtb, and activity on human IMPDH2 isoform. One of the hits, 13, a 5-amidophthalide derivative, has shown growth inhibitory potential and target specificity against the Mtb-GuaB2 enzyme. The hit, 13, is a promising molecule with potential for further development as an antitubercular agent.     

Newer human inosine 5′-monophosphate dehydrogenase 2 (hIMPDH2) inhibitors as potential anticancer agents

Human inosine 5′-monophosphate dehydrogenase 2 (hIMPDH2), being an age-old target, has attracted attention recently for anticancer drug development. Mycophenolic acid (MPA), a well-known immunosuppressant drug, was used a lead structure to design and develop modestly potent and selective analogues. The steep structure–activity relationship (SAR) requirements of the lead molecule left little scope to synthesise newer analogues. Here, newer MPA amides were designed, synthesised and evaluated for hIMPDH2 inhibition and cellular efficacy in breast, prostate and glioblastoma cell lines. Few title compounds exhibited cellular activity profile better than MPA itself. The observed differences in the overall biological profile could be attributed to improved structural and physicochemical properties of the analogues over MPA. This is the first report of the activity of MPA derivatives in glioblastoma, the most aggressive brain cancer.     

Design,synthesis, and biological evaluation of novel selective peptide inhibitors of 11β-hydroxysteroid dehydrogenase 1

The enzyme 11β-HSD1 plays a crucial role in the tissue-specific regulation of cortisol levels and it has been associated with various diseases. Inhibition of 11β-HSD1 is an attractive intervention strategy and the discovery of novel selective 11β-HSD1 inhibitors is of high relevance. In this study, we identified and evaluated a new series of selective peptide 11β-HSD1 inhibitors with potential for skin care applications. This novel scaffold was designed with the aid of molecular modeling and two previously reported inhibitors. SAR optimization yielded highly active peptides (IC₅₀ below 400?nM) that were inactive at 1?µM concentration against structurally related enzymes (11β-HSD2, 17β-HSD1 and 17β-HSD2). The best performing peptides inhibited the conversion of cortisone into cortisol in primary human keratinocytes and the most active compound, 5d, was further shown to reverse cortisone-induced collagen damage in human ex-vivo tissue.     

Design,synthesis and biological evaluation of novel dual inhibitors of acetylcholinesterase and β-secretase

To explore novel effective drugs for the treatment of Alzheimer’s disease (AD), a series of dual inhibitors of acetylcholineterase (AChE) and β-secretase (BACE-1) were designed based on the multi-target-directed ligands strategy. Among them, inhibitor 28 exhibited good dual potency in enzyme inhibitory potency assay (BACE-1: IC₅₀ = 0.567 μM; AChE: IC₅₀ = 1.83 μM), and also showed excellent inhibitory effects on Aβ production of APP transfected HEK293 cells (IC₅₀ = 98.7 nM) and mild protective effect against hydrogen peroxide (H₂O₂)-induced PC12 cell injury. Encouragingly, intracerebroventricular injection of 28 into amyloid precursor protein (APP) transgenic mice caused a 29% reduction of Aβ_1–40 production. Therefore, 28 was demonstrated as a good lead compound for the further study and more importantly, the strategy of AChE and BACE-1 dual inhibitors might be a promising direction for developing novel drugs for AD patients.     

Design,synthesis and biological evaluation of metronidazole–thiazole derivatives as antibacterial inhibitors

A series of metronidazole–thiazole derivatives has been designed, synthesized and evaluated as potential antibacterial inhibitors. All the synthesized compounds were determined by elemental analysis, ¹H NMR and MS. They were also tested for antibacterial activity against Escherichia coli, Bacillus thuringiensis, Bacillus subtilis and Pseudomonas aeruginosa as well as for the inhibition to FabH. The results showed that compound 5e exhibited the most potent inhibitory activity against E. coli FabH with IC₅₀ of 4.9 μM. Molecular modeling simulation studies were performed in order to predict the biological activity of proposed compounds. Toxicity assay of compounds 5a, 5b, 5d, 5e, 5g and 5i showed that they were noncytotoxic against human macrophage. The results revealed that these compounds offered remarkable viability.     

Cloning,characterization and validation of inosine 5′-monophosphate dehydrogenase of Babesia gibsoni as molecular drug target

The inosine monophosphate dehydrogenase (IMPDH) enzyme has been characterized and validated as a molecular drug target in other apicomplexans but not in the genus Babesia. Subsequently, we cloned and expressed a Babesia gibsoni IMPDH (BgIMPDH) cDNA in Escherichia coli. We also determined the inhibitory effect of mycophenolic acid (MPA) on recombinant BgIMPDH (rBgIMPDH) activity and the Babesia-growths in vitro. The translated BgIMPDH peptide contained thirteen amino acid residues responsible for substrate and cofactor binding in its catalytic domain with Gly374 in BgIMPDH being replaced by Ser388 in mammalian IMPDH. The native BgIMPDH enzyme in the parasite was approximately 54-kDa a mass similar to His-tag rBgIMPDH protein. The K_m values of the rBgIMPDH were 8.18 ± 0.878 (mean ± standard error of the mean) μM and 360.80 ± 43.41 μM for IMP and NAD⁺, respectively. MPA inhibited the rBgIMPDH activity yielding a K_i value of 20.93 ± 1.83 μM with respect to NAD⁺. For Babesia growths, the IC₅₀s were 0.95 ± 0.21 and 2.88 ± 0.49 μM for B. gibsoni and B. bovis, respectively. Therefore, our results suggest that MPA may inhibit the replication of Babesia parasites by targeting IMPDH enzyme of the purine pathway.     

Hybrid α-bromoacryloylamido chalcones. Design,synthesis and biological evaluation

Research into the anti-tumor properties of chalcones has received significant attention over the last few years Two novel large series of α-bromoacryloylamido chalcones 1a–m and 2a–k containing a pair of Michael acceptors in their structures, corresponding to the α-bromoacryloyl moiety and the α,β-unsaturated ketone system of the chalcone framework, were synthesized and evaluated for antiproliferative activity against five cancer cell lines. Such hybrid derivatives demonstrated significantly increased anti-tumor activity compared with the corresponding amino chalcones. The most promising lead molecules were 1k, 1m and 2j, which had the highest activity toward the five cell lines. Flow cytometry with K562 cells showed that the most active compounds resulted in a large proportion of the cells entering in the apoptotic sub-G0–G1 peak. Moreover, compound 1k induced apoptosis through the mitochondrial pathway and activated caspase-3.     

Design,synthesis and biological evaluation of 2-methyl-(1,1′-biphenyl)-pyrimidine conjugates

Small molecule inhibitors of biphenyl structure as core backbone have shown a significant effect on PD-1/PD-L1 axis, and 2-amino-pyrimidine structure is a promising privileged scaffold in medicinal chemistry and drug discovery. We designed by combination principles and synthesized 27 novel compounds with N-((2-methyl-[1,1′-biphenyl]-3-yl)methyl)pyrimidin-2-amine as a basic skeletal structure, and their anti-cancer activity was evaluated. Among compounds, 15a-d and 16b displayed strong anti-cancer effects on 9 tested cancer cell lines, in particular, the 16b did the highest inhibitive activity, but against HepG2 cells, and possessed the lowest IC₅₀ value of 2.08 μΜ towards HT-29 cells.     

Design,synthesis, and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitors—Part 2

Peripherally selective inhibition of noradrenaline reuptake is a novel mechanism for the treatment of stress urinary incontinence to overcome adverse effects associated with central action. Herein, we describe our medicinal chemistry approach to discover peripheral-selective noradrenaline reuptake inhibitors to avert the risk of P-gp-mediated DDI at the blood–brain barrier. We observed that steric shielding of the hydrogen-bond acceptors and donors (HBA and HBD) of compound 1 reduced the multidrug resistance protein 1 (MDR1) efflux ratio; however, the resulting compound 6, a methoxyacetamide derivative, was mainly metabolized by CYP2D6 and CYP2C19 in the in vitro phenotyping study, implying the risk of PK variability based on the genetic polymorphism of the CYPs. Replacement of the hydrogen atom with a deuterium atom in a strategic, metabolically hot spot led to compound 13, which was mainly metabolized by CYP3A4. To our knowledge, this study represents the first report of the effect of deuterium replacement for a major metabolic enzyme. The compound 13, N-{[(6S,7R)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-yl]methyl}-2-[(2H³)methyloxy]acetamide hydrochloride, which exhibited peripheral NET selective inhibition at tested doses in rats, increased urethral resistance in a dose-dependent manner.     

Design,synthesis and biological evaluation of pyridone–aminal derivatives as MNK1/2 inhibitors

Excessive phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) plays a major role in the dysregulation of mRNA translation and the activation of tumor cell signaling. eIF4E is exclusively phosphorylated by mitogen-activated protein kinase interacting kinases 1 and 2 (MNK1/2) on Ser209. So, MNK1/2 inhibitors could decrease the level of p-eIF4E and regulate tumor-associated signaling pathways. A series of pyridone–aminal derivatives were synthesized and evaluated as MNK1/2 inhibitors. Several compounds exhibited great inhibitory activity against MNK1/2 and selected compounds showed moderate to excellent anti-proliferative potency against hematologic cancer cell lines. In particular, compound 42i (MNK1 IC₅₀?=?7.0?nM; MNK2 IC₅₀?=?6.1?nM) proved to be the most potent compound against TMD-8 cell line with IC₅₀ value of 0.91?μM. Furthermore, 42i could block the phosphorylation level of eIF4E in CT-26 cell line, and 42i inhibited the tumor growth of CT-26 allograft model significantly. These results indicated that compound 42i was a promising MNK1/2 inhibitor for the potent treatment of colon cancer.     

Design,synthesis and biological evaluation of novel coumarin thiazole derivatives as α-glucosidase inhibitors

A new series of coumarin thiazole derivatives 7a-7t were synthesized, characterized by ¹H NMR, ¹³C NMR and element analysis, evaluated for their α-glucosidase inhibitory activity. The majority of the screened compounds displayed potent inhibitory activities with IC₅₀ values in the range of 6.24 ± 0.07–81.69 ± 0.39 μM, when compared to the standard acarbose (IC₅₀ = 43.26 ± 0.19 μM). Structure–activity relationship (SAR) studies suggest that the pattern of substitution in the phenyl ring is closely related to the biological activity of this class of compounds. Among all the tested molecules, compound 7e (IC₅₀ = 6.24 ± 0.07 μM) was found to be the most active compound in the library of coumarin thiazole derivatives. Enzyme kinetic studies showed that compound 7e is a non-competitive inhibitor with a K_i of 6.86 μM. Furthermore, the binding interactions of compound 7e with the active site of α-glucosidase were confirmed through molecular docking. This study has identified a new class of potent α-glucosidase inhibitors for further investigation.     

Design,synthesis, X-ray studies,and biological evaluation of novel macrocyclic HIV-1 protease inhibitors involving the P1′-P2′ ligands

Design, synthesis, and evaluation of a new class of HIV-1 protease inhibitors containing diverse flexible macrocyclic P1′-P2′ tethers are reported. Inhibitor 5a with a pyrrolidinone-derived macrocycle exhibited favorable enzyme inhibitory and antiviral activity (K_i = 13.2 nM, IC₅₀ = 22 nM). Further incorporation of heteroatoms in the macrocyclic skeleton provided macrocyclic inhibitors 5m and 5o. These compounds showed excellent HIV-1 protease inhibitory (K_i = 62 pM and 14 pM, respectively) and antiviral activity (IC₅₀ = 5.3 nM and 2.0 nM, respectively). Inhibitor 5o also remained highly potent against a DRV-resistant HIV-1 variant.     

Design,synthesis and biological evaluation of novel 3,4,5-trisubstituted aminothiophenes as inhibitors of p53–MDM2 interaction. Part 2

Five series of novel 3,4,5-trisubstituted aminothiophene derivatives and analogs were designed and synthesized based on our previous studies. All target compounds were evaluated for their p53–MDM2 binding inhibitory activities and anti-proliferation activities against A549 and PC3 tumor cell lines. Twelve compounds displayed comparable p53–MDM2 binding inhibitory activities to that of Nutlin-3. Among them, compound 7a exhibited marked binding affinity (IC₅₀ = 0.086 μM). In addition, most target compounds showed potent anti-proliferation activities with IC₅₀ values at low micromolar level. A good selective profile for wild-type p53 expression cell line was also observed. Molecular docking analysis was performed as well to predict possible binding modes of target compounds with MDM2.     

Design, synthesis and biological evaluation of novel imidazopyridines as potential antidiabetic GSK3β inhibitors

Design, synthesis and biological evaluation of the imidazopyridine analogs as novel GSK3β inhibitors for treatment of type 2 diabetes mellitus are described. Most of the analogs exhibited excellent inhibitory activities (IC50<44 nM) against glycogen synthase kinase 3β (GSK3β). The structure-activity relationship (SAR) of the imidazopyridine analogs and the binding mode of analog 23 in the catalytic domain of GSK3β, based on our X-ray crystallography study, are described. In particular, analog 28, which was selected as a potential drug candidate for treatment of type 2 diabetes mellitus, exhibited excellent GSK3β inhibition, pharmacokinetic profiles and blood glucose lowering effect in mouse.     

Design, synthesis and biological evaluation of β-carboline derivatives as novel inhibitors targeting B-Raf kinase

β-Carboline family of compounds is a large group of alkaloids widely distributed in nature and exhibits broad-spectrum anti-tumor activities. We designed and synthesized two series of novel 1-carboxamide- and 6-sulfonamide-substituted β-carboline derivatives 7a-p and 12a-b, and their wild type B-Raf kinase inhibitory activities were described. Most compounds showed moderate to excellent inhibitory activities. Among them, 1-carboxamide-6-(N-(3-(dimethylamino)propyl)-sulfamoyl)-β-carboline, 7e exhibited potent activity (IC(50)=1.62 μM), showing the potential for further investigation as a lead compound.     

Design,synthesis and biological evaluation of theophylline containing variant acetylene derivatives as α-amylase inhibitors

A novel pharmacophore with theophylline and acetylene moieties was constructed by using a fragment-based drug design and a series of twenty theophylline containing acetylene conjugates were designed and synthesized, and all the compounds were evaluated by enzyme-based in vitro α-amylase inhibition activity. The in vitro evaluation revealed that most of the compounds displayed good inhibitory activities, and among them nine analogs 13–15, 20, 21 and 24–27 were exhibited more or nearly as equipotent inhibitory activity with IC₅₀ values 1.11 ± 0.07, 1.14 ± 0.17, 1.07 ± 0.01 and 1.21 ± 0.03, 1.33 ± 0.09, 1.17 ± 0.01, 1.05 ± 0.02, 1.61 ± 0.04, 1.02 ± 0.03 μM respectively, as compared with standard, acarbose 1.37 ± 0.26 μM. Further, molecular docking simulation studies were done to identify the interactions and binding mode of synthesized analogs at binding site of α-amylase enzyme (PBD ID: 4GQR). Among the synthesized analogs, two compounds 25 and 27 were selected on the basis of α-amylase inhibition activity and evaluated for in vivo anti-diabetic activity by High Fat Diet-Streptozotocin (HFD-STZ) model in normal rats. At the dose of 10 mg/kg, bw, po these compounds have significantly reduced Plasma Glucose level in rats as compared to pioglitazone. The anti-diabetic activity results showed that the animal treated with the compounds 25 and 27 could better reverse and control the progression of the disease compared to the standard.     

Active-site modification of native and mutant forms of inosine 5′-monophosphate dehydrogenase from Escherichia coli K12

IMP dehydrogenase of Escherichia coli was irreversibly inactivated by Cl-IMP (6-chloro-9-beta-d-ribofuranosylpurine 5'-phosphate, 6-chloropurine ribotide). The inactivation reaction showed saturation kinetics. 6-Chloropurine riboside did not inactivate the enzyme. Inactivation by Cl-IMP was retarded by ligands that bind at the IMP-binding site. Their effectiveness was IMP>XMP>GMP>AMP. NAD(+) did not protect the enzyme from modification. Inactivation of IMP dehydrogenase was accompanied by a change in lambda(max.) of Cl-IMP from 263 to 290nm, indicating formation of a 6-alkylmercaptopurine nucleotide. The spectrum of 6-chloropurine riboside was not changed by IMP dehydrogenase. With excess Cl-IMP the increase in A(290) with time was first-order. Thus it appears that Cl-IMP reacts with only one species of thiol at the IMP-binding site of the enzyme: 2-3mol of Cl-IMP were bound per mol of IMP dehydrogenase tetramer. Of ten mutant enzymes from guaB strains, six reacted with Cl-IMP at a rate similar to that for the native enzyme. The interaction was retarded by IMP. None of the mutant enzymes reacted with 6-chloropurine riboside. 5,5'-Dithiobis-(2-nitrobenzoic acid), iodoacetate, iodoacetamide and methyl methanethiosulphonate also inactivated IMP dehydrogenase. Reduced glutathione re-activated the methanethiolated enzyme, and 2-mercaptoethanol re-activated the enzyme modified by Cl-IMP. IMP did not affect the rate of re-activation of methanethiolated enzyme. Protective modification indicates that Cl-IMP, methyl methanethiosulphonate and iodoacetamide react with the same thiol groups in the enzyme. This is also suggested by the low incorporation of iodo[(14)C]acetamide into Cl-IMP-modified enzyme. Hydrolysis of enzyme inactivated by iodo[(14)C]acetamide revealed radioactivity only in S-carboxymethylcysteine. The use of Cl-IMP as a probe for the IMP-binding site of enzymes from guaB mutants is discussed, together with the possible function of the essential thiol groups.     

Design,synthesis, and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitors – Part 3

Peripheral-selective inhibition of noradrenaline reuptake is a novel mechanism for the treatment of stress urinary incontinence to overcome adverse effects associated with central action. Here, we describe our medicinal chemistry approach to discover a novel series of highly potent, peripheral-selective, and orally available noradrenaline reuptake inhibitors with a low multidrug resistance protein 1 (MDR1) efflux ratio by cyclization of an amide moiety and introduction of an acidic group. We observed that the MDR1 efflux ratio was correlated with the pK_a value of the acidic moiety. The resulting compound 9 exhibited favorable PK profiles, probably because of the effect of intramolecular hydrogen bond, which was supported by a its single-crystal structure. The compound 9, 1-{[(6S,7R)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-yl]methyl}-2-oxo-1,2-dihydropyridine-3-carboxylic acid hydrochloride, which exhibited peripheral NET-selective inhibition at tested doses in rats by oral administration, increased urethral resistance in a dose-dependent manner.