Autoimmune central nervous system disorders: Antibody testing and its clinical utility.

A rapidly expanding repertoire of neural antibody biomarkers exists for autoimmune central nervous system (CNS) disorders. Following clinical recognition of an autoimmune CNS disorder, the detection of a neural antibody facilitates diagnosis and informs prognosis and management. This review considers the phenotypes, diagnostic assay methodologies, and clinical utility of neural antibodies in autoimmune CNS disorders. Autoimmune CNS disorders may present with a diverse range of clinical features. Clinical phenotype should inform the neural antibodies selected for testing via the use of phenotype-specific panels. Both serum and cerebrospinal fluid (CSF) are preferred in the vast majority of cases but for some analytes either CSF (e.g. N-methyl-D-aspartate receptor [NMDA-R] IgG) or serum (e.g. aquaporin-4 [AQP4] IgG) specimens may be preferred. Screening using 2 methods is recommended for most analytes, particularly paraneoplastic antibodies. We utilize murine tissue-based indirect immunofluorescence assay (TIFA) with subsequent confirmatory protein-specific testing. The cellular location of the target antigen informs choice of confirmatory diagnostic assay (e.g. blot for intracellular antigens such as Hu; cell-based assay for cell surface targets such as leucine-rich glioma inactivated 1 [LGI1]). Titers of positive results have limited diagnostic utility with the exception of glutamic acid decarboxylase (GAD) 65 IgG autoimmunity, which is associated with neurological disease at higher values. While novel antibodies are typically discovered using established techniques such as TIFA and immunoprecipitation-mass spectrometry, more recent high-throughput molecular technologies (such as protein microarray and phage-display immunoprecipitation sequencing) may expedite the process of antibody discovery. Individual neural antibodies inform the clinician regarding the clinical associations, oncological risk stratification and tumor histology, the likely prognosis, and immunotherapy choice. In the era of neural antibody biomarkers for autoimmune CNS disorders, access to appropriate laboratory assays for neural antibodies is of critical importance in the diagnosis and management of these disorders.

Final analysis of 379 pregnancy outcomes after exposure to dimethyl fumarate in a prospective international registry.

BACKGROUND: Dimethyl fumarate (DMF) has a favorable benefit-risk profile treating people with multiple sclerosis and should be used in pregnant women only if the potential benefits outweigh potential risks to the fetus. OBJECTIVE: Assess pregnancy outcomes in a completed international registry (TecGistry) of women with MS exposed to DMF. METHODS: TecGistry included pregnant women with MS exposed to DMF, with data collected at enrollment, 6-7 months gestation, 4 weeks after estimated due date, and at postpartum weeks 4, 12, and 52. Outcomes included live births, gestational size, pregnancy loss, ectopic/molar pregnancies, birth defects, and infant/maternal death. RESULTS: Of 397 enrolled, median (range) age was 32 years (19-43). Median (range) gestational week at enrollment was 10 (0-39) and at first DMF exposure was 1 (0-13). Median (range) duration of gestational DMF exposure was 5 weeks (0-40). Fifteen (3.8%) spontaneous abortions occurred. Of 360 (89.1%) live births, 323 were full term and 37 were premature. One neonatal death and no maternal deaths occurred. Adjudicator-confirmed EUROCAT birth defects were found in 2.2%. CONCLUSION: DMF exposure during pregnancy did not adversely affect pregnancy outcomes; birth defects, preterm birth, and spontaneous abortion were in line with rates from the general population.

Paraneoplastic Neurologic Disorders.

PURPOSE OF REVIEW: To provide an overview and highlight recent updates in the field of paraneoplastic neurologic disorders. RECENT FINDINGS: The prevalence of paraneoplastic neurologic disorders is greater than previously reported and the incidence has been rising over time, due to improved recognition in the era of antibody biomarkers. Updated diagnostic criteria that are broadly inclusive and also contain diagnostic risk for clinical presentations (high and intermediate) and diagnostic antibodies (high, intermediate, and low) have replaced the original 2004 criteria. Antibody biomarkers continue to be characterized (e.g., KLHL-11 associated with seminoma in men with brainstem encephalitis). Some paraneoplastic antibodies also provide insight into likely immunotherapy response and prognosis. The rise of immune checkpoint inhibitors as cancer therapeutics has been associated with newly observed immune-mediated adverse effects including paraneoplastic neurological disorders. The therapeutic approach to paraneoplastic neurologic disorders is centered around cancer care and trials of immune therapy. The field of paraneoplastic neurologic disorders continues to be advanced by the identification of novel antibody biomarkers which have diagnostic utility, and give insight into likely treatment responses and outcomes.

Synthesis and Characterization of NUC-7738, an Aryloxy Phosphoramidate of 3'-Deoxyadenosine, as a Potential Anticancer Agent.

3'-Deoxyadenosine (3'-dA, Cordycepin, 1) is a nucleoside analogue with anticancer properties, but its clinical development has been hampered due to its deactivation by adenosine deaminase (ADA) and poor cellular uptake due to low expression of the human equilibrative transporter (hENT1). Here, we describe the synthesis and characterization of NUC-7738 (7a), a 5'-aryloxy phosphoramidate prodrug of 3'-dA. We show in vitro evidence that 7a is an effective anticancer drug in a panel of solid and hematological cancer cell lines, showing its preferential cytotoxic effects on leukemic stem cells. We found that unlike 3'-dA, the activity of 7a was independent of hENT1 and kinase activity. Furthermore, it was resistant to ADA metabolic deactivation. Consistent with these findings, 7a showed increased levels of intracellular 3'-deoxyadenosine triphosphate (3'-dATP), the active metabolite. Mechanistically, levels of intracellular 3'-dATP were strongly associated with in vitro potency. NUC-7738 is now in Phase II, dose-escalation study in patients with advanced solid tumors.

Skin care advice to patients with multiple sclerosis on Fingolimod treatment at increased risk of skin malignancy-room for improvement?

Fingolimod is used to treat relapsing-remitting multiple sclerosis. It has an immunosuppressive effect that predisposes to skin malignancies. The Summary of Product Characteristics recommends that persons receiving Fingolimod be educated regarding photoprotection and vigilance of skin lesions and should undergo a dermatological evaluation at initiation of treatment and 6-12 monthly thereafter. The incidence of keratinocytic carcinomas in those on long-term immunosuppression following solid organ transplantation is declining. This trend coincided with temporal changes in immunosuppressive protocols and the introduction of skin cancer prevention programmes suggesting that the risk of developing these malignancies may be mitigated by the provision of education to patients amongst other measures. The aim of our study was to assess if health care professionals are explaining skin advice and documenting the discussion when prescribing Fingolimod in a University Hospital outpatient setting. Clinical records of consecutive patients on Fingolimod were reviewed. Data on demographics, documented provision of advice on skin protection and who provided the advice was collected. Fifty patients with multiple sclerosis were identified. Median age was 40.5 years (range 25-63). Forty-two were female (42/50, 84%). Provision of advice regarding skin protection was documented in 20% (10/50). This was provided by nurse specialists in 14% (7/50), doctors in 10% (5/50) and both in 4% (2/50). The risk of developing skin cancers can be reduced by the adoption of simple preventative measures; patients on Fingolimod are at an increased risk of developing these cancers. This study demonstrates a need for improvement in the documentation of advice around skin protection.

Interim Analysis of Pregnancy Outcomes After Exposure to Dimethyl Fumarate in a Prospective International Registry.

BACKGROUND AND OBJECTIVES: Oral delayed-release dimethyl fumarate (DMF) is not recommended during pregnancy and should only be used if the potential benefit justifies the potential fetal risk. Although DMF was well tolerated in clinical trials with consistent safety results in postmarketing surveillance, data are limited in pregnant women. The objective was to provide pregnancy outcomes and DMF exposure information from an interim analysis from a prospective, international registry (TecGistry; NCT01911767). METHODS: Women exposed to DMF from the first day of their last menstrual period before conception or during pregnancy were evaluated. Data were obtained at enrollment; 6-7 months' gestation; 4 weeks after estimated due date; and 4, 12, and 52 weeks after birth. Outcomes included live births, gestational size, pregnancy loss, birth defects, and infant or maternal death after delivery. Outcomes were analyzed cumulatively from October 30, 2013 (the start of TecGistry), to April 8, 2020. RESULTS: Of 345 enrolled patients, median (range) age was 32 (20-43) years. The mean (SD) duration of gestational weeks of DMF exposure was 4.9 (3.8). Most infants were full-term at birth (n = 249/274; 91%) and of average gestational size (n = 190/232; 82%). Of 351 outcomes, 277 were live births; 17 (5%) spontaneous abortions (95% confidence interval [CI] 2.6%-7.1%), including 1 (<1%) molar and 1 (<1%) ectopic pregnancy, were reported. There were 8 (2.9% [95% CI 1.3%-5.6%]) adjudicator-confirmed birth defects among the 277 live births. DISCUSSION: Interim results from this large registry indicate that early DMF exposure was not significantly associated with adverse pregnancy outcomes. Outcomes are consistent with previous smaller reports and with the general population. TRIAL REGISTRATION INFORMATION: TecGistry; clinical trial registration number: NCT01911767.

Vaccine hesitancy among people with multiple sclerosis.

BACKGROUND: The current severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic has raised awareness of vaccine hesitancy. Specific reasons for vaccine hesitancy among people with multiple sclerosis (pwMS) have not been fully described. Notably, pwMS may experience higher morbidity from vaccine-preventable diseases such as influenza, pneumococcal disease, and human papillomavirus (HPV)-associated warts and malignancies. Furthermore, screening for immunity against measles, mumps and rubella (MMR) is not standard practice, despite a resurgence of measles and mumps outbreaks in Europe and worldwide. We aimed to evaluate general vaccination status among pwMS to better inform vaccine practices in this cohort. METHODS: This was a prospective audit of pwMS attending an Irish tertiary referral MS centre. We designed a questionnaire that explored awareness, uptake, and hesitancy for the influenza, pneumococcal, SARS-CoV-2, HPV, and MMR vaccines. The clinician administered the questionnaire during the outpatient MS clinic. RESULTS: One-hundred-and-five pwMS participated in the audit, mean (SD) age 47.3 (12.8) years, mean MS disease duration 14.1 (9.5) years, median Expanded Disability Severity Scale (EDSS) score 2.0 (IQR 1.0-6.0), forty-nine (46.7%) were taking either maintenance immunosuppressive or immune reconstitution therapies. SARS-CoV-2 vaccine willingness among pwMS was higher (90.5 vs 60-80%) than that reported in other Western countries, and higher than that for the influenza and pneumococcal vaccines (∼80%) for which perceived unnecessity and unfamiliarity respectively were the main limiting factors. The primary reason for SARS-CoV-2 vaccine hesitancy was safety concern. PwMS who were explicitly advised by a healthcare professional to obtain the influenza vaccine were more likely to do so than those who were not (odds ratio, 8.1, 95% CI 2.8 - 23.4, p<0.001). Of pwMS currently receiving B-cell therapy (ocrelizumab/rituximab, n=12), all but one (n=11, 91.7%) have never received the pneumococcal vaccine, and a quarter (n=3) were uncertain whether to obtain this in the future. Patient-reported uptake of HPV (1.0%) and MMR (51.4%) vaccines were suboptimal. Prevalence of vaccine promotion among healthcare professionals was low (influenza vaccine, 4.8 - 32.4%; pneumococcal vaccine, 0 - 18.1%). CONCLUSIONS: Vaccine hesitancy is common (10-20%) in pwMS, consequent to insufficient knowledge and misconceptions about vaccination among pwMS and suboptimal vaccine promotion by healthcare professionals who manage pwMS. Conscientious and context-specific vaccination counselling is necessary to tackle vaccine hesitancy among pwMS, including (i) avoiding infection-associated disability accrual during MS relapses, (ii) reducing the potentially higher risk of life-threatening/treatment-refractory complications that may be observed in those who develop vaccine-preventable infections while receiving certain DMTs, and (iii) avoiding attenuated vaccine responses or delayed/interrupted DMT with early pre-treatment vaccine delivery where possible.

A clinical, molecular genetics and pathological study of a FTDP-17 family with a heterozygous splicing variant c.823-10G>T at the intron 9/exon 10 of the MAPT gene.

We report the first clinical-radiological-genetic-molecular-pathological study of a kindred with c.823-10G>T MAPT intronic variant (rs63749974) associated with frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We describe the clinical spectrum within this family and emphasize the association between MAPT gene variants and motor neuron disease. This report of a second family with FTDP-17 associated with c.823-10G>T MAPT variant strongly supports pathogenicity of the variant and confirms it is a 4-repeat (4R) tauopathy. This intronic point mutation, probably strengthens the polypyrimidine tract and alters the splicing of exon 10 (10 nucleotides into intron 9) close to the 3' splice site.

Single Diastereomers of the Clinical Anticancer ProTide Agents NUC-1031 and NUC-3373 Preferentially Target Cancer Stem Cells In Vitro.

A 3'-protected route toward the synthesis of the diastereomers of clinically active ProTides, NUC-1031 and NUC-3373, is described. The in vitro cytotoxic activities of the individual diastereomers were found to be similar to their diastereomeric mixtures. In the KG1a cell line, NUC-1031 and NUC-3373 have preferential cytotoxic effects on leukemic stem cells (LSCs). These effects were not diastereomer-specific and were not observed with the parental nucleoside analogues gemcitabine and FUDR, respectively. In addition, NUC-1031 preferentially targeted LSCs in primary AML samples and cancer stem cells in the prostate cancer cell line, LNCaP. Although the mechanism for this remains incompletely resolved, NUC-1031-treated cells showed increased levels of triphosphate in both LSC and bulk tumor fractions. As ProTides are not dependent on nucleoside transporters, it seems possible that the LSC targeting observed with ProTides may be caused, at least in part, by preferential accumulation of metabolized nucleos(t)ide analogues.

Synthesis, biological evaluation and X-ray analysis of bicalutamide sulfoxide analogues for the potential treatment of prostate cancer.

The androgen receptor (AR) is a pivotal target for the treatment of prostate cancer (PC) even when the disease progresses toward androgen-independent or castration-resistant forms. In this study, a series of sulfoxide derivatives were prepared and their antiproliferative activity evaluated in vitro against four different human prostate cancer cell lines (22Rv1, DU-145, LNCaP and VCap). Bicalutamide and enzalutamide were used as positive controls. Compound 28 displayed significant enhancement in anticancer activity across the four PC cell lines with IC50 = 9.09 - 31.11 µM compared to the positive controls: bicalutamide (IC50 = 45.20 -51.61 µM) and enzalutamide (IC50 = 11.47 - 53.04 µM). Sulfoxide derivatives of bicalutamide were prepared efficiently from the corresponding sulfides using only one equivalent of mCPBA, limiting the reaction time to 15-30 min and maintaining the temperature at 0 °C. Interestingly, three pairs of sulfoxide diastereomers were separated and NMR comparison of their diastereotopic methylene (CH2) group is presented. X-ray diffraction crystal structure analysis provided relative configuration assignment at the chiral sulfur and carbon centres. Molecular modelling study of the four diastereoisomers of compound 28 is described.

Moyamoya disease and moyamoya syndrome in Ireland: patient demographics, mode of presentation and outcomes of EC-IC bypass surgery.

BACKGROUND: There are no previously published reports regarding the epidemiology and characteristics of moyamoya disease or syndrome in Ireland. AIMS: To examine patient demographics, mode of presentation and the outcomes of extracranial-intracranial bypass surgery in the treatment of moyamoya disease and syndrome in Ireland. METHODS: All patients with moyamoya disease and syndrome referred to the National Neurosurgical Centre during January 2012-January 2019 were identified through a prospective database. Demographics, clinical presentation, radiological findings, surgical procedures, postoperative complications and any strokes during follow-up were recorded. RESULTS: Twenty-one patients were identified. Sixteen underwent surgery. Median age at diagnosis was 19 years. Fifteen were female. Mode of presentation was ischaemic stroke in nine, haemodynamic TIAs in eight, haemorrhage in three and incidental in one. Sixteen patients had Moyamoya disease, whereas five patients had moyamoya syndrome. Surgery was performed on 19 hemispheres in 16 patients. The surgical procedures consisted of ten direct (STA-MCA) bypasses, five indirect bypasses and four multiple burr holes. Postoperative complications included ischaemic stroke in one patient and subdural haematoma in one patient. The median follow-up period in the surgical group was 52 months; there was one new stroke during this period. Two patients required further revascularisation following recurrent TIAs. One patient died during follow-up secondary to tumour progression associated with neurofibromatosis type 1. CONCLUSIONS: Moyamoya is rare but occurs in Caucasians in Ireland. It most commonly presents with ischaemic symptoms. Surgical intervention in the form of direct and indirect bypass is an effective treatment in the majority of cases.

Synthesis and Biological Evaluation of Bicalutamide Analogues for the Potential Treatment of Prostate Cancer.

The androgen receptor (AR) is a pivotal target for the treatment of prostate cancer (PC) even when the disease progresses toward androgen-independent or castration-resistant forms. In this study, a series of 15 bicalutamide analogues (sulfide, deshydroxy, sulfone, and O-acetylated) were prepared and their antiproliferative activity evaluated against four different human prostate cancer cell lines (22Rv1, DU-145, LNCaP, and VCap). Bicalutamide and enzalutamide were used as positive controls. Seven of these compounds displayed remarkable enhancement in anticancer activity across the four PC cell lines. The deshydroxy analogue (16) was the most active compound with IC50 = 6.59-10.86 µM. Molecular modeling offers a plausible explanation of the higher activity of the sulfide analogues compared to their sulfone counterparts.

Drug repurposing: phosphate prodrugs of anticancer and antiviral FDA-approved nucleosides as novel antimicrobials.

OBJECTIVES: Following a drug repurposing approach, we aimed to investigate and compare the antibacterial and antibiofilm activities of different classes of phosphate prodrugs (HepDirect, cycloSal, SATE and mix SATE) of antiviral and anticancer FDA-approved nucleoside drugs [zidovudine (AZT), floxouridine (FUDR) and gemcitabine (GEM)] against a variety of pathogenic Gram-positive and -negative bacteria. METHODS: Ten prodrugs were synthesized and screened for antibacterial activity against seven Gram-negative and two Gram-positive isolates fully susceptible to traditional antibiotics, alongside six Gram-negative and five Gram-positive isolates with resistance mechanisms. Their ability to prevent and eradicate biofilms of different bacterial pathogens in relation to planktonic growth inhibition was also evaluated, together with their effect on proliferation, viability and apoptosis of different eukaryotic cells. RESULTS: The prodrugs showed decreased antibacterial activity compared with the parent nucleosides. cycloSal-GEM-monophosphate (MP) prodrugs 20a and 20b were the most active agents against Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) and retained their activity against antibiotic-resistant isolates. cycloSal-FUDR-MP 21a partially retained good activity against the Gram-positive bacteria E. faecalis, Enterococcus faecium and S. aureus. Most of the prodrugs tested displayed very potent preventive antibiofilm specific activity, but not curative. In terms of cytotoxicity, AZT prodrugs did not affect apoptosis or cell viability at the highest concentration tested, and only weak effects on apoptosis and/or cell viability were observed for GEM and FUDR prodrugs. CONCLUSIONS: Among the different prodrug approaches, the cycloSal prodrugs appeared the most effective. In particular, cycloSal (17a) and mix SATE (26) AZT prodrugs combine the lowest cytotoxicity with high and broad antibacterial and antibiofilm activity against Gram-negative bacteria.

Radiosynthesis of [18F]-Labelled Pro-Nucleotides (ProTides).

Phosphoramidate pro-nucleotides (ProTides) have revolutionized the field of anti-viral and anti-cancer nucleoside therapy, overcoming the major limitations of nucleoside therapies and achieving clinical and commercial success. Despite the translation of ProTide technology into the clinic, there remain unresolved in vivo pharmacokinetic and pharmacodynamic questions. Positron Emission Tomography (PET) imaging using [18F]-labelled model ProTides could directly address key mechanistic questions and predict response to ProTide therapy. Here we report the first radiochemical synthesis of [18F]ProTides as novel probes for PET imaging. As a proof of concept, two chemically distinct radiolabelled ProTides have been synthesized as models of 3'- and 2'-fluorinated ProTides following different radiosynthetic approaches. The 3'-[18F]FLT ProTide was obtained via a late stage [18F]fluorination in radiochemical yields (RCY) of 15-30% (n = 5, decay-corrected from end of bombardment (EoB)), with high radiochemical purities (97%) and molar activities of 56 GBq/µmol (total synthesis time of 130 min.). The 2'-[18F]FIAU ProTide was obtained via an early stage [18F]fluorination approach with an RCY of 1-5% (n = 7, decay-corrected from EoB), with high radiochemical purities (98%) and molar activities of 53 GBq/µmol (total synthesis time of 240 min).

A new series of bicalutamide, enzalutamide and enobosarm derivatives carrying pentafluorosulfanyl (SF5) and pentafluoroethyl (C2F5) substituents: Improved antiproliferative agents against prostate cancer.

SAR studies on bicalutamide, enobosarm and enzalutamide analogues, functionalised with polyfluorinated groups, is presented. Among the novel bicalutamide and enobosarm derivatives synthesised, several displayed significantly improved in vitro anticancer activity, with IC50 values in the low micromolar range against four different prostate cancer cell lines (LNCaP, VCaP, DU-145 and 22Rv1), showing up to 48-fold increase in comparison with the parent structures. In particular, SF5 enobosarm analogues were found to be most potent compounds, full AR antagonists and with favourable ADME properties. The most promising compound (48a) was evaluated for its in vivo efficacy in PC xenograft mouse model (22Rv1) with results comparable to the standard-of-care docetaxel.

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.

IRAK1 Limits TLR3/4- and IFNAR-Driven IL-27 Production through a STAT1-Dependent Mechanism.

IL-27 is a cytokine exerting pleiotropic immunomodulatory effects on a broad spectrum of immune cells. Optimal IL-27 production downstream of TLR3/4 ligand stimulation relies on autocrine type I IFN signaling, defining a first and second phase in IL-27 production. This work shows that IL-1 receptor-associated kinase 1 (IRAK1) limits TLR3/4- and IFNAR-induced IL-27 production. At the mechanistic level, we identified IRAK1 as a novel regulator of STAT1, IRF1, and IRF9. We found hyperactivation of STAT1 together with increased nuclear levels of IRF1 and IRF9 in IRAK1-deficient murine macrophages compared with control cells following stimulation with LPS and poly(I:C). IRAK1-deficient human microglial cells showed higher basal levels of STAT1 and STAT2 compared with control cells. Blocking the kinase activity of TBK1/IKKε in IRAK1 knockdown human microglial cells reduced the high basal levels of STAT1/2, uncovering a TBK1/IKKε kinase-dependent mechanism controlling basal levels of STAT1/2. Stimulating IRAK1 knockdown human microglial cells with IFN-ß led to increased IL-27p28 expression compared with control cells. In IRAK1-deficient murine macrophages, increased IL-27 levels were detected by ELISA following IFN-ß stimulation compared with control macrophages together with increased nuclear levels of p-STAT1, IRF1, and IRF9. Treatment of wild-type and IRAK1-deficient murine macrophages with fludarabine similarly reduced TLR3/4-induced IL-27 cytokine levels. To our knowledge, this work represents the first report placing IRAK1 in the IFNAR pathway and identifies IRAK1 as an important regulator of STAT1, controlling IL-27 production downstream of TLR3/4 and IFNAR signaling pathways.

Synthesis and biological evaluation of 6-substituted-5-fluorouridine ProTides.

A new family of thirteen phosphoramidate prodrugs (ProTides) of different 6-substituted-5-fluorouridine nucleoside analogues were synthesized and evaluated as potential anticancer agents. In addition, antiviral activity against Chikungunya (CHIKV) virus was evaluated using a cytopathic effect inhibition assay. Although a carboxypeptidase Y assay supported a putative mechanism of activation of ProTides built on 5-fluorouridine with such C6-modifications, the Hint docking studies revealed a compromised substrate-activity for the Hint phosphoramidase-type enzyme that is likely responsible for phosphoramidate bioactivation through P-N bond cleavage and free nucleoside 5'-monophosphate delivery. Our observations may support and explain to some extent the poor in vitro biological activity generally demonstrated by the series of 6-substituted-5-fluorouridine phosphoramidates (ProTides) and will be of guidance for the design of novel phosphoramidate prodrugs.

Synthetic Approaches for the Preparation of Phosphoramidate Prodrugs of 2'-Deoxypseudoisocytidine.

A synthetic procedure for the preparation of phosphoramidate prodrugs of C-nucleosides is reported. Different phosphorochloridates were reacted with 3'-O-protected N-acetyl-2'-deoxypseudoisocytidine or 3'-O-protected 2'-deoxypseudoisocytidine, followed by acidic hydrolysis of the protecting group. In the presence of the N-acetyl moiety, the enolisable keto group of the nucleobase was able to react (like the 5'-OH) with the phosphorochloridates to give bisphosphorylated derivatives. Epimerisation (ß to α) occurred if the amino group of the nucleobase was unprotected. These side reactions demonstrate the peculiar behaviour of C-nucleosides compared to their nucleoside analogues. It was demonstrated that the first enzymatic activation step for this new class of prodrugs can be mediated by carboxypeptidase and that it follows the same pathway and rate reported for ProTides of more conventional nucleoside analogues. These new phosphoramidate derivatives deserve further investigation for their therapeutic potential as anti-cancer agents.