Synthesis and Evaluation of Prodrugs of α-Carboxy Nucleoside Phosphonates.

A range of lipophilic prodrugs of α-carboxy nucleoside phosphonates, potent inhibitors of HIV-1 reverse transcriptase without requiring prior phosphorylation, were synthesized to evaluate their in vivo potency against HIV in cell culture. A series of prodrug derivatives bearing a free carboxylic acid where the phosphonate was masked with bispivaloyloxymethyl, diisopropyloxycarbonyloxymethyl, bisamidate, aryloxyphosphoramidate, hexadecyloxypropyl, CycloSal, and acycloxybenzyl moieties were synthesized, adapting existing methodologies for phosphonate protection to accommodate the adjacent carboxylic acid moiety. The prodrugs were assayed for anti-HIV activity in CEM cell cultures─the bispivaloyloxymethyl free acid monophosphonate prodrug exhibited some activity (inhibitory concentration-50 (IC50) 59 ± 17 µM), while the other prodrugs were inactive at 100 µM. A racemic bispivaloyloxymethyl methyl ester monophosphonate prodrug was also prepared to assess the suitability of the methyl ester as a carboxylic acid prodrug. This compound exhibited no activity against HIV in cellular assays.

Enantiocontrolled Preparation of ϒ-Substituted Cyclohexenones: Synthesis and Kinase Activity Assays of Cyclopropyl-Fused Cyclohexane Nucleosides.

The enantioselective preparation of the two isomers of 4-hydroxy-2-cyclohexanone derivatives 1a,b was achieved, starting from a common cyclohexenone, through asymmetric transfer hydrogenation (ATH) reactions using bifunctional ruthenium catalysts. From these versatile intermediates, a stereoselective route to a cytosine analogue built on a bicyclo [4.1.0]heptane scaffold is described. Nucleoside kinase activity assays with this cyclopropyl-fused cyclohexane nucleoside, together with other related nucleosides (2a-e), were performed, showing that thymine- and guanine- containing compounds have affinity for herpes simplex virus Type 1 (HSV-1) thymidine kinase (TK) but not for human cytosolic TK-1, thus pointing to their selectivity for herpetic TKs but not cellular TKs.

Benzothiazole Derivatives as Multifunctional Antioxidant Agents for Skin Damage: Structure-Activity Relationship of a Scaffold Bearing a Five-Membered Ring System.

Skin diseases often give multifactorial damages; therefore, the development of multifunctional compounds represents a suitable approach especially against disorders that are induced by oxidative stress. Thus, taking into account the successful results we achieved on benzimidazoles, we have devised a new series of isosteric benzothiazoles and investigated their antioxidant, photoprotective, antifungal and antiproliferative activity. Particular attention has been paid to synergistic antioxidant and photoprotective properties. For compounds 9a and 10a, a multifunctional profile was outlined, supported by an excellent filtering capacity, mainly UVB, which has higher capacities than those of the reference PBSA which is currently in the market as a UV sunscreen filter. The two compounds were also the best in terms of growth inhibition of dermatophytes and Candida albicans, and 10a also showed good antioxidant activity. Furthermore, 9a was also effective on melanoma tumor cells (SK-Mel 5), making these compounds good candidates in the development of new skin protective and preventive agents.

γ-Non-Symmetrically Dimasked TriPPPro Prodrugs as Potential Antiviral Agents against HIV.

Nucleoside analogue reverse transcriptase inhibitors (NRTI) and nucleoside analogue monophosphate prodrugs are used in combination antiretroviral therapy (cART). The design of antivirally active nucleoside triphosphate prodrugs is a recent and an important advancement in the field of nucleoside analogue drug development. Here, we report on TriPPPro-derivatives of nucleoside analogue triphosphates (NTPs) that comprised two different acyloxybenzyl-masks at the γ-phosphate of the NTP aiming to achieve the metabolic bypass. Thus, γ-non-symmetrically dimasked TriPPPro-compounds (γ-(AB,ab)-d4TTPs) were synthesized and they proved to be active against HIV-1 and HIV-2 in cultures of infected wild-type human CD4+ T-lymphocyte (CEM/0) cells and more importantly also in thymidine kinase-deficient CD4+ T-cells (CEM/TK-). From hydrolysis studies both in phosphate buffer (PB, pH 7.3) and CEM cell extracts, there was surprisingly no differentiation in the cleavage of the two acyloxybenzyl prodrug-masks. However, if within one of the two acyloxybenzyl groups a short PEG-type methoxytriglycol group was introduced, the "standard" acyloxybenzyl-mask was cleaved with high preference.

γ-Ketobenzyl-Modified Nucleoside Triphosphate Prodrugs as Potential Antivirals.

The antiviral activity of nucleoside reverse transcriptase inhibitors is often hampered by insufficient phosphorylation. Nucleoside triphosphate analogues are presented, in which the γ-phosphate was covalently modified by a non-bioreversible, lipophilic 4-alkylketobenzyl moiety. Interestingly, primer extension assays using human immunodeficiency virus reverse transcriptase (HIV-RT) and three DNA-polymerases showed a high selectivity of these γ-modified nucleoside triphosphates to act as substrates for HIV-RT, while they proved to be nonsubstrates for DNA-polymerases α, ß, and γ. In contrast to d4TTP, the γ-modified d4TTPs showed a high resistance toward dephosphorylation in cell extracts. A series of acyloxybenzyl-prodrugs of these γ-ketobenzyl nucleoside triphosphates was prepared. The aim was the intracellular delivery of a stable γ-modified nucleoside triphosphate to increase the selectivity of such compounds to act in infected versus noninfected cells. Delivery of γ-ketobenzyl-d4TTPs was proven in T-lymphocyte cell extracts. The prodrugs were potent inhibitors of HIV-1/2 in cultures of infected CEM/0 cells and more importantly in thymidine kinase-deficient CD4+ T-cells.

Skin Damages-Structure Activity Relationship of Benzimidazole Derivatives Bearing a 5-Membered Ring System.

In the search for scaffolds for multifunctional compounds we investigated the structure activity relationship of a class of benzimidazole derivatives bearing 5-membered ring. The newly synthesized and the already known compounds were divided into three classes that present different substituent at 5 position of the benzimidazole ring (-H, -COOH or -SO3H) and different heterocycle at position 2 (thiophene, furan or pyrrole). All the derivatives were synthesized and tested to determine their photoprotective profile against UV rays, in vitro antioxidant capacity against different radicals (DPPH and FRAP test), antifungal inhibitory activity (dermatophytes and Candida albicans), antiviral and antiproliferative activity. A Structure-Activity Relationship study indicated compound 10, bearing a pyrrole heterocycle on the benzimidazole ring, as the best multifunctional derivative of the series and as potential candidate for the development of drugs especially in case of melanoma.

Water-soluble fullerene-based nanostructures with promising antiviral and myogenic activity.

Here we report a straightforward method for the synthesis of a water-soluble C60 fullerene derivative decorated with five residues of phosphonic acid. Self-assembly of the synthesized compound in aqueous solution leads to the formation of nanostructures with unprecedented myogenic and antiviral activity.

Design, synthesis and evaluation of benzothiazole derivatives as multifunctional agents.

Oxidative stress is the product or aetiology of various multifactorial diseases; on the other hand, the development of multifunctional compounds is a recognized strategy for the control of complex diseases. To this end, a series of benzothiazole derivatives was synthesized and evaluated for their multifunctional effectiveness as antioxidant, sunscreen (filter), antifungal and antiproliferative agents. Compounds were easily synthesized via condensation reaction between 2-aminothiophenols and different benzaldehydes. SAR study, particularly in position 2 and 6 of benzothiazoles, led to the identification of 4g and 4k as very interesting potential compounds for the design of multifunctional drugs. In particular, compound 4g is the best blocker of hERG potassium channels expressed in HEK 293 cells exhibiting 60.32% inhibition with IC50 = 4.79 µM.

Prodrugs of γ-Alkyl-Modified Nucleoside Triphosphates: Improved Inhibition of HIV Reverse Transcriptase.

The development of nucleoside triphosphate prodrugs is one option to apply nucleoside reverse transcriptase inhibitors. Herein, we report the synthesis and evaluation of d4TTP analogues, in which the γ-phosphate was modified covalently by lipophilic alkyl residues, and acyloxybenzyl prodrugs of these γ-alkyl-modified d4TTPs, with the aim of delivering of γ-alkyl-d4TTP into cells. Selective formation of γ-alkyl-d4TTP was proven with esterase and in CD4+ -cell extracts. In contrast to d4TTP, γ-alkyl-d4TTPs proved highly stable against dephosphorylation. Primer extension assays with HIV reverse transcriptase (RT) and DNA-polymerases α, ß or γ showed that γ-alkyl-d4TTPs were substrates for HIV-RT only. In antiviral assays, compounds were highly potent inhibitors of HIV-1 and HIV-2 also in thymidine-kinase-deficient T-cell cultures (CEM/TK- ). Thus, the intracellular delivery of such γ-alkyl-nucleoside triphosphates may potentially lead to nucleoside triphosphates with a higher selectivity towards the viral polymerase that can act in virus-infected cells.

Synthesis and Biological Evaluation of New Antitubulin Agents Containing 2-(3',4',5'-trimethoxyanilino)-3,6-disubstituted-4,5,6,7-tetrahydrothieno[2,3-c]pyridine Scaffold.

Two novel series of compounds based on the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine and 4,5,6,7-tetrahydrobenzo[b]thiophene molecular skeleton, characterized by the presence of a 3',4',5'-trimethoxyanilino moiety and a cyano or an alkoxycarbonyl group at its 2- or 3-position, respectively, were designed, synthesized, and evaluated for antiproliferative activity on a panel of cancer cell lines and for selected highly active compounds, inhibition of tubulin polymerization, and cell cycle effects. We have identified the 2-(3',4',5'-trimethoxyanilino)-3-cyano-6-methoxycarbonyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine derivative 3a and its 6-ethoxycarbonyl homologue 3b as new antiproliferative agents that inhibit cancer cell growth with IC50 values ranging from 1.1 to 4.7 µM against a panel of three cancer cell lines. Their interaction with tubulin at micromolar levels leads to the accumulation of cells in the G2/M phase of the cell cycle and to an apoptotic cell death. The cell apoptosis study found that compounds 3a and 3b were very effective in the induction of apoptosis in a dose-dependent manner. These two derivatives did not induce cell death in normal human peripheral blood mononuclear cells, suggesting that they may be selective against cancer cells. Molecular docking studies confirmed that the inhibitory activity of these molecules on tubulin polymerization derived from binding to the colchicine site.

Design, synthesis and biological evaluation of 2-alkoxycarbonyl-3-anilinoindoles as a new class of potent inhibitors of tubulin polymerization.

A new class of inhibitors of tubulin polymerization based on the 2-alkoxycarbonyl-3-(3',4',5'-trimethoxyanilino)indole molecular skeleton was synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization and cell cycle effects. The results presented show that the methoxy substitution and location on the indole nucleus plays an important role in inhibition of cell growth, and the most favorable position for the substituent was at C-6. In addition, a small-size ester function (methoxy/ethoxycarbonyl) at the 2-position of the indole core was desirable. Also, analogues that were alkylated with methyl, ethyl or n-propyl groups or had a benzyl moiety on the N-1 indolic nitrogen retained activity equivalent to those observed in the parent N-1H analogues. The most promising compounds of the series were 2-methoxycarbonyl-3-(3',4'.5'-trimethoxyanilino)-5-methoxyindole 3f and 1-methyl-2-methoxycarbonyl-3-(3',4'.5'-trimethoxyanilino)-6-methoxy-indole 3w, both of which target tubulin at the colchicine site with antitubulin activities comparable to that of the reference compound combretastatin A-4.

Direct arylation of C60Cl6 and C70Cl8 with carboxylic acids: a synthetic avenue to water-soluble fullerene derivatives with promising antiviral activity.

We report unprecedented Friedel-Crafts arylation of chlorofullerenes C60Cl6 and C70Cl8 with unprotected carboxylic acids as an efficient single-step synthesis of the inherently stable water-soluble fullerene derivatives. Using this method, a series of previously unaccessible compounds was obtained without chromatographic purification in almost quantitative yields. Promising anti-HIV activity comparable to characteristics of commercial drugs was demonstrated for some of these compounds.

Potent antiviral activity of carbohydrate-specific algal and leguminous lectins from the Brazilian biodiversity.

Brazil has one of the largest biodiversities in the world. The search for new natural products extracted from the Brazilian flora may lead to the discovery of novel drugs with potential to treat infectious and other diseases. Here, we have investigated 9 lectins extracted and purified from the Northeastern Brazilian flora, from both leguminous species: Canavalia brasiliensis (ConBr), C. maritima (ConM), Dioclea lasiocarpa (DLasiL) and D. sclerocarpa (DSclerL), and algae Amansia multifida (AML), Bryothamniom seaforthii (BSL), Hypnea musciformis (HML), Meristiella echinocarpa (MEL) and Solieria filiformis (SfL). They were exposed to a panel of 18 different viruses, including HIV and influenza viruses. Several lectins showed highly potent antiviral activity, often within the low nanomolar range. DSclerL and DLasiL exhibited EC50 values (effective concentration of lectin required to inhibit virus-induced cytopathicity by 50%) of 9 nM to 46 nM for HIV-1 and respiratory syncytial virus (RSV), respectively, DLasiL also inhibited feline corona virus at an EC50 of 5 nM, and DSclerL, ConBr and ConM showed remarkably low EC50 values ranging from 0.4 to 6 nM against influenza A virus strain H3N2 and influenza B virus. For HIV, evidence pointed to the blockage of entry of the virus into its target cells as the underlying mechanism of antiviral action of these lectins. Overall, the most promising lectins based on their EC50 values were DLasiL, DSclerL, ConBr, ConM, SfL and HML. These novel findings indicate that lectins from the Brazilian flora may provide novel antiviral compounds with therapeutic potential.

Alpha-carboxynucleoside phosphonates: direct-acting inhibitors of viral DNA polymerases.

Acyclic nucleoside phosphonates represent a well-defined class of clinically used nucleoside analogs. All acyclic nucleoside phosphonates need intracellular phosphorylation before they can bind viral DNA polymerases. Recently, a novel class of alpha-carboxynucleoside phosphonates have been designed to mimic the natural 2'-deoxynucleotide 5'-triphosphate substrates of DNA polymerases. They contain a carboxyl group in the phosphonate moiety linked to the nucleobase through a cyclic or acyclic bridge. Alpha-carboxynucleoside phosphonates act as viral DNA polymerase inhibitors without any prior requirement of metabolic conversion. Selective inhibitory activity against retroviral reverse transcriptase and herpesvirus DNA polymerases have been demonstrated. These compounds have a unique mechanism of inhibition of viral DNA polymerases, and provide possibilities for further modifications to optimize and fine tune their antiviral DNA polymerase spectrum.

Dolutegravir Monotherapy of Simian Immunodeficiency Virus-Infected Macaques Selects for Several Patterns of Resistance Mutations with Variable Virological Outcomes.

Drug resistance remains a major concern for human immunodeficiency virus (HIV) treatment. To date, very few resistance mutations have emerged in first-line combination therapy that includes the integrase strand transfer inhibitor (INSTI) dolutegravir (DTG). In vitro, DTG selects for several primary mutations that induce low-level DTG resistance; secondary mutations, while increasing the level of resistance, however, further impair replication fitness, which raised the idea that DTG monotherapy may be feasible. The simian immunodeficiency virus (SIV) rhesus macaque model of HIV infection can be useful to explore this concept. Nine macaques were infected with virulent SIVmac251 and started on DTG monotherapy during either acute (n = 2) or chronic infection (n = 7). Within 4 weeks of treatment, all animals demonstrated a reduction in viremia of 0.8 to 3.5 log RNA copies/ml plasma. Continued treatment led to overall sustained benefits, but the outcome after 10 to 50 weeks of treatment was highly variable and ranged from viral rebound to near pretreatment levels to sustained suppression, with viremia being 0.5 to 5 logs lower than expected based on pretreatment viremia. A variety of mutations previously described to confer low-level resistance of HIV-1 to DTG or other INSTI were detected, and these were sometimes followed by mutations believed to be compensatory. Some mutations, such as G118R, previously shown to severely impair the replication capacity in vitro, were associated with more sustained virological and immunological benefits of continued DTG therapy, while other mutations, such as E92Q and G140A/Q148K, were associated with more variable outcomes. The observed variability of the outcomes in macaques warrants avoidance of DTG monotherapy in HIV-infected people.IMPORTANCE A growing number of anti-HIV drug combinations are effective in suppressing virus replication in HIV-infected persons. However, to reduce their cost and risk for toxicity, there is considerable interest in simplifying drug regimens. A major concern with single-drug regimens is the emergence of drug-resistant viral mutants. It has been speculated that DTG monotherapy may be a feasible option, because DTG may have a higher genetic barrier for the development of drug resistance than other commonly used antiretrovirals. To explore treatment initiation with DTG monotherapy, we started SIV-infected macaques on DTG during either acute or chronic infection. Although DTG initially reduced virus replication, continued treatment led to the emergence of a variety of viral mutations previously described to confer low-level resistance of HIV-1 to DTG, and this was associated with variable clinical outcomes. This unpredictability of mutational pathways and outcomes warns against using DTG monotherapy as initial treatment for HIV-infected people.

Novel Conjugated Unsaturated Ketones with Submicromolar Potencies Towards some Leukemic and Colon Cancer Cells.

BACKGROUND: Cancer continues to be the major health burden worldwide. There is an urgent need for the development of novel antineoplastic compounds to treat this devastating condition. Various alkylating anticancer drugs have been employed in the clinic for treating cancers. Unsaturated conjugated ketones are a group of alkylators which are of significant interest as potent antineoplastic agents. OBJECTIVE: The goal of this study is to discover unsaturated conjugated ketones which are novel potent cytotoxins displaying growth-inhibitory properties towards neoplasms and also to serve as cytotoxic warheads in drug development. METHODS: A variety of 3,5-bis (benzylidene)-4-piperidones 2a-n were synthesized and evaluated against a number of neoplastic cell lines. The short-term neurotoxicity of 2a-k, n was evaluated in mice by i.p. administration using doses level of 30, 100 and 300 mg/kg. Statistical correlations for determining structure-activity relationships were performed using an SPSS software. RESULTS: A number of compounds display cytotoxic potencies in the region of 10-7 to 10-8 M and some of the structural features contributing to the cytotoxicity were identified. Compounds 2a-d, 2h demonstrated substantially higher cytotoxic potencies compared to melphalan and 5- fluorouracil against a panel of leukemic and colon cancer cell lines. These lead cytotoxins comply with drug-likeness properties. In general, the antineoplastics 2 are well tolerated in mice using a short-term neurotoxicity screening. CONCLUSION: In general, this group of compounds comprises excellent cytotoxic agents, which warrant their further development as cytotoxic warheads.

Synthesis and Antiviral Activity of Water-Soluble Polycarboxylic Derivatives of [60]Fullerene Loaded with 3,4-Dichlorophenyl Units.

We have synthesized a series of water-soluble polycarboxylic derivatives of [60]fullerene with a gradually changed polarity by combining three to five polar (ionic) malonate addends with two to zero hydrophobic dichlorobenzene units and explored their antiviral activity. It has been shown that decreasing the number of the ionogenic carboxylic groups in the molecules enhanced their antiviral activity against HIV-1 and suppressed their action against HIV-2. The obtained results implied that the charged states and hydrophobicity of the water-soluble polycarboxylic fullerene derivatives affect significantly their biological properties.

Symmetrical Diamidates as a Class of Phosphate Prodrugs to Deliver the 5'-Monophosphate Forms of Anticancer Nucleoside Analogues.

The application of phosphorodiamidate technology to pyrimidine and purine nucleosides with anticancer activity to potentially overcome the resistance mechanisms associated with parent nucleosides is reported. Sixteen symmetrical phosphorodiamidates were prepared from the natural amino acids l-alanine and glycine. All the compounds were evaluated for their cytotoxic activity against a wide panel of solid and leukaemic tumour cell lines. In addition, a carboxypeptidase Y assay was performed on a representative phosphorodiamidate in order to reveal the putative bioactivation pathway for the reported phosphorodiamidate-type prodrugs.

Synthesis of Guanine α-Carboxy Nucleoside Phosphonate (G-α-CNP), a Direct Inhibitor of Multiple Viral DNA Polymerases.

The synthesis of guanine α-carboxy nucleoside phosphonate (G-α-CNP) is described. Two routes provide access to racemic G-α-CNP 9, one via base construction and the other utilizing Tsuji-Trost allylic substitution. The latter methodology was also applied to the enantiopure synthesis of both antipodes of G-α-CNP, each of which showing interesting antiviral DNA polymerase activity. Additionally, we report an improved multigram scale preparation of the cyclopentene building block 10, starting material for the preferred Tsuji-Trost route to 9.

Design, synthesis, in vitro antiproliferative activity and apoptosis-inducing studies of 1-(3',4',5'-trimethoxyphenyl)-3-(2'-alkoxycarbonylindolyl)-2-propen-1-one derivatives obtained by a molecular hybridisation approach.

Inhibition of microtubule function using tubulin targeting agents has received growing attention in the last several decades. The indole scaffold has been recognized as an important scaffold in the design of novel compounds acting as antimitotic agents. Indole-based chalcones, in which one of the aryl rings was replaced by an indole, have been explored in the last few years for their anticancer potential in different cancer cell lines. Eighteen novel (3',4',5'-trimethoxyphenyl)-indolyl-propenone derivatives with general structure 9 were synthesized and evaluated for their antiproliferative activity against a panel of four different human cancer cell lines. The highest IC50 values were obtained against the human promyelocytic leukemia HL-60 cell line. This series of chalcone derivatives was characterized by the presence of a 2-alkoxycarbonyl indole ring as the second aryl system attached at the carbonyl of the 3-position of the 1-(3',4',5'-trimethoxyphenyl)-2-propen-1-one framework. The structure-activity relationship (SAR) of the indole-based chalcone derivatives was investigated by varying the position of the methoxy group, by the introduction of different substituents (hydrogen, methyl, ethyl or benzyl) at the N-1 position and by the activity differences between methoxycarbonyl and ethoxycarbonyl moieties at the 2-position of the indole nucleus. The antiproliferative activity data of the novel synthesized compounds revealed that generally N-substituted indole analogues exhibited considerably reduced potency as compared with their parent N-unsubstituted counterparts, demonstrating that the presence of a hydrogen on the indole nitrogen plays a decisive role in increasing antiproliferative activity. The results also revealed that the position of the methoxy group on the indole ring is a critical determinant of biological activity. Among the synthesized derivatives, compound 9e, containing the 2-methoxycarbonyl-6-methoxy-N-1H-indole moiety exhibited the highest antiproliferative activity, with IC50 values of 0.37, 0.16 and 0.17 µM against HeLa, HT29 and MCF-7 cancer cell lines, respectively, and with considerably lower activity against HL-60 cells (IC50: 18 µM). This derivative also displayed cytotoxic properties (IC50 values ∼1 µM) in the human myeloid leukemia U-937 cell line overexpressing human Bcl-2 (U-937/Bcl-2) via cell cycle progression arrest at the G2-M phase and induction of apoptosis. The results obtained also demonstrated that the antiproliferative activity of this molecule is related to inhibition of tubulin polymerisation. The presence of a methoxy group at the C5- or C6-position of the indole nucleus, as well as the absence of substituents at the N-1-indole position, contributed to the optimal activity of the indole-propenone-3',4',5'-trimethoxyphenyl scaffold.