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d-cycloserine is a broad-spectrum antibiotic that is currently being used as a secondary choice in the treatment of tuberculosis. In recent years, it has become more popular, due to its effect on the nervous system. In this current study, we provide evidence that The International Pharmacopoeia HPLC–UV method for d-cycloserine impurity profiling is not repeatable due to the variable response of cycloserine dimer, one of d-cycloserine impurities. Therefore, we introduced the DOSY (diffusion ordered spectroscopy) NMR (nuclear magnetic resonance) technique to determine the levels of d-cycloserine impurities in pharmaceutical dosage forms. The DOSY NMR technique allowed separation of d-cycloserine, its degradation products, and key process impurities in concentrations below pharmacopoeial specification limits. The proposed DOSY NMR method allowed accurate identification and quantification of the cycloserine dimer, which was not possible through the use of the pharmacopoeial HPLC method. The current method has the potential for practical use in analytical laboratories of the pharmaceutical industry.

Due to their ability to inhibit viral DNA or RNA replication, nucleoside analogues have been used for decades as potent antiviral therapeutics. However, one of the major limitations of nucleoside analogues is the development of antiviral resistance. In that regard, flexible nucleoside analogues known as “fleximers” have garnered attention over the years due to their ability to survey different amino acids in enzyme binding sites, thus overcoming the potential development of antiviral resistance. Acyclic fleximers have previously demonstrated antiviral activity against numerous viruses including Middle East Respiratory Syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), and, most recently, flaviviruses such as Dengue (DENV) and Yellow Fever Virus (YFV). Due to these interesting results, a Structure Activity Relationship (SAR) study was pursued in order to analyze the effect of the pyrimidine functional group and acyl protecting group on antiviral activity, cytotoxicity, and conformation. The results of those studies are presented herein.

In the present paper, we report on the synthesis, and in vitro antiviral and cytostatic activities of a series of novel imidazole[4,5-e][1,3]diazepine-4,8-dione (compounds 9–11) and acyclic carbamoyl imino-ureido imidazole (compounds 12 and 13) derivatives. These new type of chemical entities showed no significant activity on the broad spectrum of DNA and RNA viruses. Results of antiproliferative assays performed on a panel of selected human tumor cell lines revealed that only compounds 1 and 5 showed moderate and selective cytostatic effect against HeLa cells (IC50 = 24 and 32 µM) with no concomitant cytotoxic effects on human normal fibroblasts (BJ). Importantly, an imidazole derivative containing a pyrrolidine moiety linked via an ethylenic spacer (3) showed a selective cytostatic effect toward cervical carcinoma (HeLa) cells (IC50 = 9.5 µM) with no apparent cytotoxicity on human normal fibroblasts (BJ). This compound can be therefore considered as a potential anti-tumor lead compound for further synthetic structure optimization.

We report on the synthesis of 4-hydroxycoumarin dimers 1–15 bearing an aryl substituent on the central linker and fused benzopyranocoumarin derivatives 16–20 and on their in vitro broad anti-DNA and RNA virus activity evaluations. The chemical identities and structure of compounds 1–20 were deduced from their homo- and heteronuclear NMR measurements whereas the conformational properties of 5, 14 and 20 were assessed by the use of 1D difference NOE enhancements. Unequivocal proof of the stereostructure of compounds 7, 9, 16 and 18 was obtained by single crystal X-ray diffraction method. The X-ray crystal structure analysis revealed that two 4-hydroxycoumarin moieties in the 4-trifluoromethylphenyl- and 2-nitrophenyl derivatives (compounds 7 and 9, respectively) are intramolecularly hydrogen-bonded between hydroxyl and carbonyl oxygen atoms. Consequently, the compounds 7 and 9 adopt conformations in which two 4-hydroxy-coumarin moieties are anti-disposed. Antiviral activity evaluation results indicated that the 4-bromobenzylidene derivative of bis-(4-hydroxycoumarin) (compound 3) possesses inhibitory activity against HSV-1 (KOS), HSV-2 (G), vaccinia virus and HSV-1 TK- KOS (ACVr) at a concentration of 9–12 μM and at a minimum cytotoxic concentration (MCC) greater than 20 μM. Compounds 4–6, 8, and 20 were active against feline herpes virus (50% effective concentration, EC50 = 5–8.1 μM), that is at a 4-7-fold lower concentration than the MCC.

Novel N-methoxymethylated (MOM) pyrimidine (4-13) and pyrimidine-2,4-diones (15-17) nucleoside mimetics in which an isobutyl side-chain is attached at the C-6 position of the pyrimidine moiety were synthesized. Synthetic methods via O-persilylated or N-anionic uracil derivatives have been evaluated for the synthesis of N-1- and/or N-3-MOM pyrimidine derivatives with C-6 acyclic side-chains. A synthetic approach using an activated N-anionic pyrimidine derivative afforded the desired N,N-1,3-diMOM and N-1-MOM pyrimidines 4 and 5 in good yield. Introduction of fluorine into the side-chain was performed with DAST as the fluorinating reagent to give a N,N-1,3-diMOM pyrimidine 13 with a 1-fluoro-3-hydroxyisobutyl moiety at C-6. Conformational study of the monotritylated N-1-MOM pyrimidine 12 by the use of the NOE experiments revealed the predominant conformation of the compound to be one where the hydroxymethyl group in the C-6 side-chain is close to the N-1-MOM moiety, while the OMTr is in proximity to the CH3-5 group. Contrary to this no NOE enhancements between the N-1-MOM group and hydroxymethyl or fluoromethyl protons in 13 were observed, which suggested a nonrestricted rotation along the C-6 side-chain. Fluorinated N,N-1,3-diMOM pyrimidine 13 emerged as a model compound for development of tracer molecules for non-invasive imaging of gene expression using positron emission tomography (PET).

The synthetic route for introduction of fluorophenylalkyl (compounds 5, 7, 14 and 15) and fluorophenylalkenyl (compounds 4E and 13) side chains at C-6 of the pyrimidine nucleus involved the lithiation of the pyrimidine derivatives 1, 2 and 11 and subsequent nucleophilic addition or substitution reactions of the organolithium intermediate thus obtained with 2-fluorophenylacetone, 4-fluoroacetophenone or ethyl 4-fluorobenzoate as electrophiles. The structures of novel compounds were confirmed by 1H-, 19F- and 13C-NMR and MS. Compounds 8 and 10 containing unsaturated fluorophenylalkyl side chains showed better inhibitory effect than their saturated fluorophenylalkylated pyrimidine counterparts 7 and 9. A conformational study based on NOE enhancements showed the importance of the double bond and substitution in the side chain for the conformational preferences in relation to inhibitory activity. Among all tested compounds, C-5 furyl (12) and phenyl (13 and 15) substituted pyrimidine derivatives showed significant cytostatic activities against all tested tumor cell lines.

A kinetic approach was used to determine the radical scavenging activities of sinapic acid and its derivatives: sinapine, 4-vinylsyringol, syringic acid, syringaldehyde, and ethyl, propyl and butyl sinapate. The responses were expressed as rates of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH˙) scavenging (RS), superoxide radical (O2˙−) scavenging (RFF), and β-carotene bleaching in the emulsion system (RB). For RS and RB, the esters of sinapic acid showed the highest responses while, for RFF, this was seen for syringic acid. The effectiveness of the selected compounds for scavenging these free radicals was also determined at a fixed endpoint. The early response parameters were demonstrated to be good discriminators in assessing differences for antioxidants with comparable fixed endpoint activity. The primary feature that ranks the kinetic data and the endpoint determinations is interpreted in terms of the mechanisms of the reactions involved in each of the assays conducted.