Anti-Inflammatory Activity of Purine Nucleoside Analogs

Abstract

It is well known that adenosine plays an important role in inflammatory processes. A collection of adenosine analogs modified in the base and/or sugar functional moiety have been synthesized and submitted for biological testing. Each purine nucleoside analog was tested for inhibition of endothelial cell activation by various inflammatory stimuli. A number of analogs exhibited potent anti-inflammatory activity. Animal studies have been carried out on AMG002370 which was found to potently inhibit adjuvant induced arthritis in the Lewis rat.     

6-Cyclopropylpurines as Novel Potent Analogs of Cytokinins

6-Alkynyl-, trans-6-alkenyl-, trans-6-cyclopropyl-and 6-alkylpurines structurally related to the cytokinin 6-benzylaminopurine (BAP) have been synthesized and examined with a radish cotyledon assay as plant growth stimulators. The growth stimulation obtained with the 6-alkylpurines trans-cyclopropylpurines was very close to that obtained with BAP, and the trans-styrylpurines were somewhat less effective. The fact that the conformationally locked cyclopropanes exhibit growth-stimulating effects comparable to the flexible 6-alkylpurines and to BAP, supports the hypothesis that the orientation of the NH-CH₂ bond in “the active conformation” of BAP is close to anti, which means that the torsion angle C(6)-N(6)-CH₂-C is approximately 180 degrees.     

Cytotoxic and Anti-Inflammatory Activity of 3,19-Isopropylidene-/Arylidene-Andrographolide Analogs

A series of 3,19-isopropylidene-/or arylidene-andrographolide analogs were synthesized and their structures were confirmed by NMR spectroscopic methodology. Twenty-five analogs were evaluated for their in vitro cytotoxic activity against HT-29, HepG2 and LNCaP cancer cell lines based on the sulforhodamine B (SRB) assay. Analog 2 f exhibited the most potent cytotoxic activity, with IC₅₀ values of 11.14 and 9.25 μM on HepG2 and LNCaP cancer cell lines, respectively. Esterification of hydroxy functional group at position C-14 in andrographolide analogs, 2 a and 2 b _, showed somewhat higher cytotoxicity than the precursor. In addition, andrographolide analogs ( 2 a – 2 d , 2 f , 3 a , 4 a and 4 h ) were evaluated for the NO inhibitory activity in the LPS stimulated RAW264.7 macrophages. The most active analog 2 a significantly reduced nitric oxide (NO) production from LPS stimulated RAW264.7 cells, with IC₅₀ values of 0.34±0.02 μM providing encouraging results for anti-inflammatory compound development.     

新型二氢嘧啶类化合物的合成及其抗乙型肝炎病毒活性研究

以芳香氰基化合物为起始原料,经胺解、乙酰化、催化氢化、关环等步骤得到新型二氢嘧啶类化合物14个,结构通过核磁共振氢谱、质谱、元素分析确证,并利用HepG2.2.15细胞进行了体外抗HBV活性评价.结果表明部分化合物具有明显的抗HBVDNA复制作用,其中化合物5c和5n抑制HBVDNA复制的半数有效浓度分别为0.87μmol/L和0.67μmol/L.初步探讨了活性化合物的构效关系,同时运用分子排阻色谱法考察了活性化合物与病毒衣壳蛋白的相互作用.     

Novel Gallate Triphenylphosphonium Derivatives with Potent Antichagasic Activity

Chagas disease is one of the most neglected tropical diseases in the world, affecting nearly 15 million people, primarily in Latin America. Only two drugs are used for the treatment of this disease, nifurtimox and benznidazole. These drugs have limited efficacy and frequently induce adverse effects, limiting their usefulness. Consequently, new drugs must be found. In this study, we demonstrated the in vitro trypanocidal effects of a series of four gallic acid derivatives characterized by a gallate group linked to a triphenylphosphonium (TPP⁺) moiety (a delocalized cation) via a hydrocarbon chain of 8, 10, 11, or 12 atoms (TPP⁺-C₈, TPP⁺-C₁₀, TPP⁺-C₁₁, and TPP⁺-C₁₂, respectively). We analyzed parasite viability in isolated parasites (by MTT reduction and flow cytometry) and infected mammalian cells using T. cruzi Y strain trypomastigotes. Among the four derivatives, TPP⁺-C₁₀ and TPP⁺-C₁₂ were the most potent in both models, with EC₅₀ values (in isolated parasites) of 1.0 ± 0.6 and 1.0 ± 0.7 μM, respectively, and were significantly more potent than nifurtimox (EC₅₀ = 4.1 ± 0.6 μM). At 1 μM, TPP⁺-C₁₀ and TPP⁺-C₁₂ induced markers of cell death, such as phosphatidylserine exposure and propidium iodide permeabilization. In addition, at 1 μM, TPP⁺-C₁₀ and TPP⁺-C₁₂ significantly decreased the number of intracellular amastigotes (TPP⁺-C₁₀: 24.3%, TPP⁺-C₁₂: 19.0% of control measurements, as measured by DAPI staining) and the parasite’s DNA load (C₁₀: 10%, C₁₂: 13% of control measurements, as measured by qPCR). Based on the previous mode of action described for these compounds in cancer cells, we explored their mitochondrial effects in isolated trypomastigotes. TPP⁺-C₁₀ and TPP⁺-C₁₂ were the most potent compounds, significantly altering mitochondrial membrane potential at 1 μM (measured by JC-1 fluorescence) and inducing mitochondrial transition pore opening at 5 μM. Taken together, these results indicate that the TPP⁺-C₁₀ and TPP⁺-C₁₂ derivatives of gallic acid are promising trypanocidal agents with mitochondrial activity.     

A Novel Time-Dependent CENP-E Inhibitor with Potent Antitumor Activity

Centromere-associated protein E (CENP-E) regulates both chromosome congression and the spindle assembly checkpoint (SAC) during mitosis. The loss of CENP-E function causes chromosome misalignment, leading to SAC activation and apoptosis during prolonged mitotic arrest. Here, we describe the biological and antiproliferative activities of a novel small-molecule inhibitor of CENP-E, Compound-A (Cmpd-A). Cmpd-A inhibits the ATPase activity of the CENP-E motor domain, acting as a time-dependent inhibitor with an ATP-competitive-like behavior. Cmpd-A causes chromosome misalignment on the metaphase plate, leading to prolonged mitotic arrest. Treatment with Cmpd-A induces antiproliferation in multiple cancer cell lines. Furthermore, Cmpd-A exhibits antitumor activity in a nude mouse xenograft model, and this antitumor activity is accompanied by the elevation of phosphohistone H3 levels in tumors. These findings demonstrate the potency of the CENP-E inhibitor Cmpd-A and its potential as an anticancer therapeutic agent.     

Synthesis and Biological Activity of Some Benzochromenoquinolinones: Tacrine Analogs as Potent Anti‐Alzheimer's Agents

Alzheimer's disease (AD) is a well‐known neurodegenerative disorder affecting millions of old people worldwide and the corresponding epidemiological data emphasize the importance of the disease. As AD is a multifactorial illness, various single target directed drugs that have reached clinical trials have failed. Therefore, various factors associated with outset of AD have been considered in targeted drug discovery. In this work, various benzochromenoquinolinones were synthesized and evaluated for their cholinesterase and BACE1 inhibitory activities as well as neuroprotective and metal‐chelating properties. Among the synthesized compounds, 14‐amino‐13‐(3‐nitrophenyl)‐2,3,4,13‐tetrahydro‐1H‐benzo[6,7]chromeno[2,3‐b]quinoline‐7,12‐dione ( 6m ) depicted the best inhibitory activity toward acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with IC₅₀s of 0.86 and 6.03 μm , respectively. Also, the compound could inhibit β‐secretase 1 (BACE1) with IC₅₀=19.60 μm and showed metal chelating ability toward Cu²⁺, Fe²⁺, and Zn²⁺. In addition, docking study demonstrated desirable interactions of compound 6m with amino acid residues characterizing AChE, BChE, and BACE1.     

Potent Inhibition of Macrophomate Synthase by Reaction Intermediate Analogs

Potent inhibitors for macrophomate synthase, which has recently been found to catalyze a highly unusual five-step chemical transformation, were explored. Among 11 oxalacetate analogs tested, only three analogs had moderate to relatively strong inhibitory activities (I ₅₀ 1.3-8.1 mM). On the other hand, among 35 bicyclic intermediate analogs synthesized, two diacids were found to be the most potent inhibitors (I ₅₀ 0.80, 0.84 mM) which had a much higher affinity than that of the natural substrate 2-pyrone. (-)-Enantiomers of the diacids showed 30 times stronger activity (I ₅₀ 0.34, 0.41 mM) than (+)-ones. The I ₅₀/K _m values (0.20, 0.24) showed their potent inhibitions. Competitive inhibitions were observed in two representative inhibitors.     

RTA 408, A Novel Synthetic Triterpenoid with Broad Anticancer and Anti-Inflammatory Activity

Semi-synthetic triterpenoids are antioxidant inflammation modulator (AIM) compounds that inhibit tumor cell growth and metastasis. Compounds in the AIM class bind to Keap1 and attenuate Nrf2 degradation. In the nucleus, Nrf2 increases antioxidant gene expression and reduces pro-inflammatory gene expression. By increasing Nrf2 activity, AIMs reduce reactive oxygen species and inflammation in the tumor microenvironment, which reverses tumor-mediated immune evasion and inhibits tumor growth and metastasis. AIMs also directly inhibit tumor cell growth by modulating oncogenic signaling pathways, such as IKKβ/NF-κB. Here, we characterized the in vitro antioxidant, anti-inflammatory, and anticancer activities of RTA 408, a novel AIM that is currently being evaluated in patients with advanced malignancies. At low concentrations (≤ 25 nM), RTA 408 activated Nrf2 and suppressed nitric oxide and pro-inflammatory cytokine levels in interferon-γ-stimulated RAW 264.7 macrophage cells. At higher concentrations, RTA 408 inhibited tumor cell growth (GI₅₀ = 260 ± 74 nM) and increased caspase activity in tumor cell lines, but not in normal primary human cells. Consistent with the direct effect of AIMs on IKKβ, RTA 408 inhibited NF-κB signaling and decreased cyclin D1 levels at the same concentrations that inhibited cell growth and induced apoptosis. RTA 408 also increased CDKN1A (p21) levels and JNK phosphorylation. The in vitro activity profile of RTA 408 is similar to that of bardoxolone methyl, which was well-tolerated by patients at doses that demonstrated target engagement. Taken together, these data support clinical evaluation of RTA 408 for cancer treatment.     

Potent Antitumor Activity Generated by a Novel Tumor Specific Cytotoxic T Cell

Hepatocellular carcinoma is one of the most common malignant neoplasms in the world and is the main cause of death in patients with liver cirrhosis. Surgical intervention is not suitable for majority of hepatocellular carcinoma. Investigation of the effective targeting to the tumor cells is essential for both primary tumors and metastases. Tumor specific cytotoxic T lymphocytes (CTL) have been considered to be the attractive vehicles for delivering therapeutic agents toward various tumor diseases. This study was to explore the distribution pattern of CTL carrying the lentiviral vectors with the characteristic of adenoviral E1 gene under the control of the cell activation-dependent CD40 ligand promoter (Lenti/hCD40L/E1AB). Following transduction with adenoviral vectors containing chimeric type 5 and type 35 fiber proteins (Ad5/35-TRAIL), these CTLs produced infectious virus when exposed to HepG2 cells. We assessed the therapeutic ability of CTLs using MTT, Western blot and colony formation assay. The novel CTL harboring Lenti/hCD40L/E1AB and Ad5/35-TRAIL caused proliferation inhibition and significant apoptosis in hepatocellular carcinoma cell lines. Thus, the novel CTL may be useful for the development of gene therapy approaches to hepatocellular carcinoma.     

Synthesis and Potent Anti-Leukemic Activity of Novel 5′-Deoxycarbocyclic C-Nucleoside Phosphonic Acids

The first synthetic route to 5′-deoxycarbocyclic C-nucleoside [9-deazaadenosine, (pyrrolo[3,2-d]pyrimidine)] phosphonic acids from commercially available 1,4-dihydroxy-2-butene was described. The key C-C bond formation from cyclopentanone to base precursor was performed using Knoevenagel-type condensation. Synthesized C-nucleoside phosphonic acids were tested for anti-HIV activity as well as anti-leukemic activity. They showed moderate cytotoxicity derived anti-HIV activity and anti-leukemic activity.     

Potent Anti-HIV Chemokine Analogs Direct Post-Endocytic Sorting of CCR5

G protein-coupled receptors (GPCRs) are desensitized and internalized following activation. They are then subjected to post-endocytic sorting (degradation, slow recycling or fast recycling). The majority of research on post-endocytic sorting has focused on the role of sequence-encoded address structures on receptors. This study focuses on trafficking of CCR5, a GPCR chemokine receptor and the principal entry coreceptor for HIV. Using Chinese Hamster Ovary cells stably expressing CCR5 we show that two different anti-HIV chemokine analogs, PSC-RANTES and 5P14-RANTES, direct receptor trafficking into two distinct subcellular compartments: the trans-Golgi network and the endosome recycling compartment, respectively. Our results indicate that a likely mechanism for ligand-directed sorting of CCR5 involves capacity of the chemokine analogs to elicit the formation of durable complexes of CCR5 and arrestin2 (beta-arrestin-1), with PSC-RANTES eliciting durable association in contrast to 5P14-RANTES, which elicits only transient association.     

6-Hydroxyflavone and Derivatives Exhibit Potent Anti-Inflammatory Activity among Mono-, Di- and Polyhydroxylated Flavones in Kidney Mesangial Cells

Inflammatory responses by kidney mesangial cells play a critical role in the glomerulonephritis. The anti-inflammatory potential of nineteen mono-, di- and polyhydroxylated flavones including fisetin, quercetin, morin, tricetin, gossypetin, apigenin and myricetin were investigated on rat mesangial cells with lipopolysaccharide (LPS) as the inflammatory stimuli. 6-Hydroxyflavone and 4′,6-dihydroxyflavone exhibited high activity with IC₅₀ in the range of 2.0 μM, a much better inhibition potential in comparison to the well-studied polyhydroxylated flavones. Interestingly, the anti-inflammatory activity was not due to direct quenching of NO radicals. Investigation on derivatives with methylation, acetylation or sulfation of 6-hydroxyl group revealed that 6-methoxyflavone was the most potent with an IC₅₀ of 192 nM. Mechanistic study indicated that the anti-inflammatory activity of 6-methoxyflavone arose via the inhibition of LPS-induced downstream inducible NO synthase in mesangial cells. The identification of 6-hydroxyflavone and 6-methoxyflavone with potent anti-inflammatory activity in kidney mesangial cells provides a new flavone scaffold and direction to develop naturally derived products for potential nephritis prevention and treatment.     

Synthesis of Novel Polycyclic Nucleoside Analogs

Abstract

We have synthesized polycyclic nucleoside derivatives by a novel, one pot procedure by reacting 4-0-TPS-pyrimidine nucleosides with aromatic diamines. The reaction is limited in scope but provides easy access to certain previously unknown heterocyclic ring systems.₂ 4-0-Triisopro- pylphenylsulfonyl-pyrimidine nucleosides were reacted with aromatic diamines leading to fused, polycyclic ring systems: o-phenylenediamine yielded the pyrimido[1,6-a]benzimidazole, 2.3- diaminonaphthalene gave the naphth[2′,3′:4,5]imidam [1.2-flpyrimidine and 1.8-diaminonaph- thalene led to the pyrimido[l,6-a]perimidine ring system. The reaction is unique because two connected nucleophilic centers react with the pyrimidine nucleoside to form an extended ring system. However, reactions of pyrimidine nucleosides with electrophiles are well known. E.g., reaction of cytidine and adenosine with bromoacetaldehyde yields ethenocytidine and ethenoadenosine) and on reaction of cytidine with 1′-methylthiaminium salts dipyrimido[1,6-a:4′,5′-d]pyrimidine derivatives are obtained.₄ Other polycyclic bases have been made from cytidine and adenosine by photochemical reactions₅.     

Anti-Inflammatory Activity of Choisya ternata Kunth Essential Oil,Ternanthranin, and Its Two Synthetic Analogs (Methyl and Propyl N-Methylanthranilates)

Choisya ternata Kunth (Rutaceae) is native to North America where it is popularly known as “Mexican orange”. In this study, the anti-inflammatory effects of the essential oil (EO) obtained from the leaves of C. ternata, one of its minor components (ternanthranin—ISOAN) and its two synthetic analogues (methyl and propyl N-methylanthranilate – MAN and PAN) were evaluated. Mice pretreated with the EO (EO) obtained from C. ternata leaves (3–100 mg/kg, p.o.), ISOAN, MAN or PAN (1–30 mg/kg, p.o.) and the reference drugs, morphine (1 mg/kg, p.o.) and acetylsalicylic acid (ASA, 100 mg/kg, p.o.), were evaluated in inflammation models such as formalin and subcutaneous air pouch models, with measurement of cell migration, exudate volume, protein extravasation, nitric oxide and pro-inflammatory cytokines. The EO from C. ternata significantly inhibited the time that the animals spent licking the formalin-injected paw in the second phase of the model at their higher doses (30 and 100 mg/kg, respectively). An inhibition of the inflammatory reaction induced after subcutaneous carrageenan injection into air pouch was also observed. In this model, the EO significantly reduced cell migration, exudate volume, protein extravased, and the increase in levels of inflammatory mediators (nitric oxide, TNF-α and IL-1β). ISOAN, MAN and PAN behaved in the same fashion at much smaller doses. Also, these molecules were able to show significant effects in the reduction of paw edema (at all tested doses) when the phlogistic agent was carrageenan, bradykinin, 5-HT, PGE2, C48/80 or 12-O-tetradecanoylphorbol-acetate (TPA). None of the tested doses had any effect in reducing histamine-induced edema. Our results indicate that the EO from C. ternata and anthranilate derivatives demonstrates an anti-inflammatory effect.     

Synthesis and Anti-Angiogenic Activity of Cortistatin Analogs

Analogs of cortistatins, a series of anti-angiogenic compounds isolated from the Indonesian marine sponge Cortisium simplex, were synthesized from estrone by using the Suzuki-Miyaura coupling reaction as the key step. The estrone-isoquinoline hybridized compound showed selective inhibitory activity against the proliferation and VEGF-induced migration of HUVEC.     

Synthesis of Novel Peptidyl Adenosine Antibiotic Analogs

A small library of peptidyl adenosine antibiotic analogs was synthesized, under the Pilot Scale Library Program of the NIH Roadmap initiative, from 2′,3′-O-isoproylideneadenosine-5′-carboxylic acid 2 in excellent yield. The coupling of the amino terminus of L-2-aminophenylbutyric methyl ester to a free 5′-carboxylic acid moiety of 2 followed by sodium hydroxide treatment led to carboxylic acid analog 4. Hydrolysis of this latter gave unprotected nucleoside analog 5. Intermediate 4 served as the precursor for the preparation of novel peptidyl adenosine analogs 6–18 in good yields and high purity through peptide coupling reactions to diverse amine derivatives. No marked anticancer and antimalaria activity was noted on preliminary cellular testing; however these analogs should be useful candidates for other types of biological activity.