Nucleoside Protection Research Articles

Targeting dihydrofolate reductase: Design, synthesis and biological evaluation of novel 6-substituted pyrrolo[2,3-d]pyrimidines as nonclassical antifolates and as potential antitumor agents

A novel series of 6-substituted pyrrolo[2,3-d]pyrimidines with reversed amide moieties from the lead compound 1a were designed and synthesized as nonclassical antifolates and as potential antitumor agents. Target compounds 1–9 were successfully obtained through two sequential condensation reactions from the key intermediate 2-amino-6-(2-aminoethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one. In preliminary antiproliferation assay, all compounds demonstrated submicromolar to nanomolar inhibitory effects against KB tumor cells, whereas compounds 1–3 also exhibited nanomolar antiproliferative activities toward SW620 and A549 cells. In particular, compounds 1–3 were significantly more potent than the positive control methotrexate (MTX) and pemetrexed (PMX) to A549 cells. The growth inhibition induced cell cycle arrest at G1-phase with S-phase suppression. Along with the results of nucleoside protection assays, inhibition assays of dihydrofolate reductase (DHFR) clearly elucidated that the intracellular target of the designed compounds was DHFR. Molecular modeling studies suggested two binding modes of the target compounds with DHFR.

Novel 6-substituted benzoyl and non-benzoyl straight chain pyrrolo[2,3-d]pyrimidines as potential antitumor agents with multitargeted inhibition of TS, GARFTase and AICARFTase

A novel series of 6-substituted benzoyl and non-benzoyl straight chain pyrrolo[2,3-d]pyrimidines were designed and synthesized as potential antitumor agents targeting both thymidylate and purine nucleotide biosynthesis. Starting from the key intermediate 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidin-6-yl-acetic acid, target compounds 1–6 were successfully obtained through two sequential condensation and saponification reactions in decent yield. The newly synthesized compounds showed antiproliferative potencies against a panel of tumor cell lines including KB, SW620 and MCF7. In particular, most compounds of this series exhibited nanomolar to subnanomolar inhibitory activities toward KB tumor cells, significantly more potent than the positive control methotrexate (MTX) and pemetrexed (PMX). Along with the results of nucleoside protection assays, molecular modeling studies suggested that the antitumor activity of compound 6 could be attributed to multitargeted inhibition of folate-dependent enzymes thymidylate synthase (TS), glycinamide ribonucleotide formyltransferase (GARFTase) and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase). Growth inhibition by compound 6 also induced distinct early apoptosis and cell cycle arrest at S-phase, which resulted in cell death.

DDQ mediated regiospecific protection of primary alcohol and deprotection under neutral conditions: Application of new p -methoxy benzyl-pixyl ether as reagent of choice for nucleoside protection

A simple and efficient protocol is described for regiosepecific protection of primary hydroxyl group both in nucleosides and other molecules with p-methoxy-benzyl 2,7-dimethyl pixylether (MBDPE) in presence of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Furthermore, swift deprotection of 2, 7-dimethylpixyl (DMPx) is accomplished with DDQ in MeOH. Both procedures are successfully implemented on gram-scale synthesis of modified nucleosides. This protocol offers mild and neutral conditions for selective protection and deprotection of DMPx group while compatible in presence of other conventional protecting groups such as benzoyl, benzyl, THP, TBDPS and acetonide.

Design, synthesis and biological evaluation of 6-substituted pyrrolo[2,3-d]pyrimidines as dual inhibitors of TS and AICARFTase and as potential antitumor agents

A new series of 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines, with an isosteric replacement of the side chain amide moiety to a sulfur atom, were designed and synthesized as multitargeted antifolates as well as potential antitumor agents. Starting from previously synthesized 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidin-6-yl-acetic acid, a reduction by lithium triethylborohydride and successive mesylation afforded the key mesylate. Nucleophilic substitution by mercaptoacetic or mercaptopropionic acid methyl esters, followed by hydrolysis and condensation with pyridinyl-methylamines provided the nonclassical compounds 1–6, whereas condensation with glutamic acid diethyl ester hydrochloride and saponification afforded the classical analogs 7–8. All target compounds exhibited inhibitory activities toward KB, SW620 and A549 tumor cell lines. The most potent compounds of this series, 7 and 8, are better inhibitors against A549 cells than methotrexate (MTX) and pemetrexed (PMX). Nucleoside protection assays establish compound 8 a dual inhibitor of thymidylate synthase (TS) and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) targeting both de novo thymidylate and purine nucleotide biosynthesis, which is further verified by the molecular modeling studies. Analogous to PMX, target compound 8 alternates the cell cycle of SW620 cells with S-phase accumulation and induces apoptosis, leading to cell death.

Abstract 5492: Study the variation of the glutamate moiety of AG94, a targeted, potent GARFTase inhibitor, as antitumor agents with the 2-amino-6-substituted pyrrolo[2,3-d]pyrimidine scaffold.

Abstract We have been extensively involved in the elaboration of substituted pyrrolo[2,3-d]pyrimidines to obtain potential targeted agents without reduced folate carrier (RFC) activity. Lack of selectivity for RFC is a major cause of significant toxicity in the clinic associated with antifolate anticancer agents. These efforts have led to the discovery of several series of potent targeted agents, which showed specificity for folate receptors (FRs) and/or the proton coupled folate transporter (PCFT). These transport systems are two targets for selective cancer chemotherapy. One such targeted agent, AG94, was reported by us as the most efficacious targeted antifolate antitumor agent with PCFT and FR selectivity over RFC. AG94 is a GARFTase inhibitor and its targeted transport and inhibition of GARFTase was demonstrated to be responsible for its selective in vitro and in vivo antitumor activity. We were interested in carrying out a structure-activity relationship study (SAR) with AG94 as the lead compound. For this study we elected the glutamate moiety of AG94 for modification to determine the effect of this variation on transport as well as cell inhibition in culture. The glutamate moiety of AG94 was replaced with various natural and unnatural amino acids or completely deleted in an attempt to vary the chain length between the two carboxylic groups and retain one or none of the carboxylic moieties in the target compounds. The results showed that AG190 was relatively potent with an IC50 of 9.0 nM in human tumor KB cells in culture. A complete set of cell proliferation assays in CHO sublines afforded a SAR study of the transporters with defined transport as well as for GARFTase inhibitory activity. With FRα expressing RT16 CHO cells, an order of decreasing sensitivities (AG94>AG189∼AG190>AG213>AG214>AG187) was obtained. For FRβ expressing D4 CHO cells, it showed that AG94>AG190>>AG189, whereas AG187, AG213 and AG214 are inactive. All target compounds are inactive in PCFT expressing R2/PCFT4 cells. In the binding assays towards PCFT and FRα with AG189 and AG190, we found that AG190 and AG189 still bound to PCFT although somewhat less avidly than that for AG94 towards PCFT, and AG189 along with AG190 exhibited very similar binding affinity towards FRα compared to AG94. In nucleoside protection assay, surprisingly neither adenosine nor AICA protect and suggested a change in intracellular target for AG189 and AG190 compared to AG94. Based on the profile of protection, we postulate that both AG189 and AG190 retain their targeted mechanism for transport but may not act as GARFTase inhibitors, a role previously established for AG94. Further study to elucitdate their exact role is currently underway. The ability to manipulate the intracellular target by variation in the glutamate moiety is novel and remarkable for this series. Citation Format: Aleem Gangjee, Sai Zhao, Lalit Kumar, Christina Cherian, Steven Orr, Jenny Huang, Zhanjun Hou, Larry H. Matherly. Study the variation of the glutamate moiety of AG94, a targeted, potent GARFTase inhibitor, as antitumor agents with the 2-amino-6-substituted pyrrolo[2,3-d]pyrimidine scaffold. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5492. doi:10.1158/1538-7445.AM2013-5492

Abstract 5494: Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate regioisomers target folate receptor alpha positive ovarian cancer cells via inhibition of de novo purine nucleotide biosynthesis.

Abstract Epithelial ovarian cancers (EOC) represent a unique treatment challenge as over 50% of patients are diagnosed with advanced stage disease when surgical and chemotherapeutic interventions prove futile. Approximately 90% of EOC are folate receptor (FR) α positive and FR levels correlate with stage and grade. FRα-targeted therapies represent a rational treatment option for patients with advanced EOC whose treatments fail. There are three primary mechanisms of folate transport in mammalian cells, the reduced folate carrier (RFC), the proton-coupled folate transporter (PCFT) and FRs. RFC is the major transporter for folates and is expressed ubiquitously in normal tissues and tumors. Hence, developing novel antifolates that bypass cellular accumulation by RFC should reduce dose-limiting toxicities. PCFT is expressed in many solid tumors including ovarian cancer and is active at acidic pH. Previous studies established that a novel 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolate with an isosteric thienoyl substitution and a three carbon bridge (AG94) showed selective cellular uptake by FRα and PCFT over RFC. [1,3]Thienoyl regioisomers (AG150 and AG154) of AG94 showed subnanomolar potencies toward both KB nasopharyngeal carcinoma (IC50s = 0.1 and 0.09 nM, respectively) and IGROV1 ovarian carcinoma (IC50s of 0.64 and 0.1nM, respectively). While AG150 and AG154 were substrates for both PCFT and FRs, like AG94, their inhibitory effects at low drug concentrations were mediated primarily via FRα, as indicated by their capacities to bind with high affinities to FRα and protection by excess folic acid. Nucleoside protection assays with adenosine and 5-amino-4-imidazole carboxamide established that the first folate-dependent enzyme in the de novo purine biosynthesis pathway, glycinamide ribonucleotide (GAR) formyltransferase (GARFT), was the primary intracellular target for AG150 and AG154. GARFT targeting was confirmed by in situ metabolic labeling with [14C]glycine and measurement of accumulated [14C]formyl GAR in KB cells, for which IC50s were 1.35 and 1.46 nM, respectively. The purine salvage enzyme methylthioadenosine phosphorylase (MTAP) is frequently co-deleted with CDKN2a in solid tumors including ovarian cancer. Growth inhibitory effects of AG150 and AG154 were reversed in the presence of methylthioadenosine in MTAP-deficient IGROV1 cells transfected with MTAP cDNA. Our results demonstrate potent anti-tumor activities of the 6-pyrrolo[2,3-d]pyrimidine thienoyl regioisomers of AG94, AG150 and AG154, associated with their FRα-mediated cellular uptake and inhibition of GARFT in de novo purine biosynthesis. Preservation of purine salvage including MTAP is likely an important cellular determinant of antitumor efficacy for this class of novel tumor-targeted agents. Citation Format: Shermaine K. Mitchell-Ryan, Lei Wang, Steven Orr, Sita Kugel, Christina Cherian, Aleem Gangjee, Larry H. Matherly. Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate regioisomers target folate receptor alpha positive ovarian cancer cells via inhibition of de novo purine nucleotide biosynthesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5494. doi:10.1158/1538-7445.AM2013-5494

Therapeutic targeting malignant mesothelioma with a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate via its selective uptake by the proton-coupled folate transporter

We examined whether the novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate, compound 2, might be an effective treatment for malignant pleural mesothelioma (MPM), reflecting its selective membrane transport by the proton-coupled folate transport (PCFT) over the reduced folate carrier (RFC). HeLa sublines expressing exclusively PCFT (R1-11-PCFT4) or RFC (R1-11-RFC6) and H2452 MPM cells were assayed for transport with [(3)H]compound 2. [(3)H]Polyglutamate metabolites of compound 2 were measured in R1-11-PCFT4 and H2452 cells. In vitro cell proliferation assays and colony formation assays were performed. Inhibition of glycinamide ribonucleotide formyltransferase (GARFTase) was assayed by nucleoside protection assays and in situ GARFTase assays with [(14)C]glycine. In vivo efficacy was established with early- and advanced-stage H2452 xenografts in severe-combined immunodeficient (SCID) mice administered intravenous compound 2. [(3)H]Compound 2 was selectively transported by PCFT and was metabolized to polyglutamates. Compound 2 selectively inhibited proliferation of R1-11-PCFT4 cells over R1-11-RFC6 cells. H2452 human MPM cells were sensitive to the antiproliferative effects of compound 2. By colony-forming assays with H2452 cells, compound 2 was cytotoxic. Compound 2 inhibited GARFTase in de novo purine biosynthesis. In vivo efficacy was confirmed toward early- and advanced-stage H2452 xenografts in SCID mice administered compound 2. Our results demonstrate potent antitumor efficacy of compound 2 toward H2452 MPM cells in vitro and in vivo, reflecting its efficient membrane transport by PCFT, synthesis of polyglutamates, and inhibition of GARFTase. Selectivity for non-RFC cellular uptake processes by tumor-targeted antifolates such as compound 2 presents an exciting new opportunity for treating solid tumors.

Convenient synthesis of nucleoside 5′-triphosphates for RNA transcription

By generating a selective phosphitylating reagent in situ, nucleoside 5'-triphosphates can be conveniently synthesized in one pot. This novel strategy without nucleoside protection has been developed to largely simplify synthesis of the nucleoside triphosphates. This demonstrated principle can be applied to the 5'-triphosphate synthesis of both native and modified nucleosides.

ChemInform Abstract: An Efficient One‐Pot N‐Acylation of Deoxy‐ and Ribo‐cytidine Using Carboxylic Acids Activated in situ with 2‐Chloro‐4,6‐dimethoxy‐1,3,5‐triazine.

: Selective acylation, Nucleoside protection, 2-Chloro-4,6-dimethoxy-1,3,5-triazine, Activated acyl group, Single-step protectionSynthetic oligonucleotides (ODNs) are an emerging class of drug molecules with a broad spectrum for therapeutic appli-cations. In the ODNs synthesis, a fundamental process is the internucleotide-bond formation achieved

An Efficient One-pot N-Acylation of Deoxy- and Ribo-cytidine Using Carboxylic Acids Activated in situ with 2-Chloro-4,6-dimethoxy-1,3,5-triazine

: Selective acylation, Nucleoside protection, 2-Chloro-4,6-dimethoxy-1,3,5-triazine, Activated acyl group, Single-step protectionSynthetic oligonucleotides (ODNs) are an emerging class of drug molecules with a broad spectrum for therapeutic appli-cations. In the ODNs synthesis, a fundamental process is the internucleotide-bond formation achieved

Preface

Preface

Protection of Nucleosides for Oligonucleotide Synthesis

Current Protocols in Nucleic Acid ChemistryVolume 39, Issue 1 p. 2.0.1-2.0.3 Introduction Protection of Nucleosides for Oligonucleotide Synthesis Serge L. Beaucage, Serge L. BeaucageSearch for more papers by this author Serge L. Beaucage, Serge L. BeaucageSearch for more papers by this author First published: 15 December 2009 https://doi.org/10.1002/0471142700.nc0200s39Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. View the latest in Nucleic Acid Chemistry This article also appears in:Protection of Nucleosides for Oligonucleotide Synthesis RelatedInformation

Protection of Nucleosides for Oligonucleotide Synthesis

Protection of Nucleosides for Oligonucleotide Synthesis

Protection of Nucleosides for Oligonucleotide Synthesis

Protection of Nucleosides for Oligonucleotide Synthesis

Fast, quantitative nucleoside protection under solvent-free conditions

Persilylation of nucleoside hydroxyls was effected in quantitative yields under solvent-free conditions using a ball mill. In addition, one-pot persilylation and acylation of cytidine was performed as an exemplar reaction demonstrating the utility of solvent-free approaches to nucleoside chemistry.

Protection of Nucleosides for Oligonucleotide Synthesis

Current Protocols in Nucleic Acid ChemistryVolume 30, Issue 1 p. 2.0.1-2.0.3 Introduction Protection of Nucleosides for Oligonucleotide Synthesis Serge L. Beaucage, Serge L. BeaucageSearch for more papers by this author Serge L. Beaucage, Serge L. BeaucageSearch for more papers by this author First published: 15 September 2007 https://doi.org/10.1002/0471142700.nc0200s30Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. View the latest in Nucleic Acid Chemistry RelatedInformation

The effect of a novel transition state inhibitor of methylthioadenosine phosphorylase on pemetrexed activity

Pemetrexed is a new-generation antifolate inhibitor of thymidylate synthase (TS) and a weaker inhibitor of glycinamide ribonucleotide transformylase (GARFT) required for de novo purine synthesis. Methylthioadenosine phosphorylase (MTAP) salvages purines by releasing adenine from methylthioadenosine and is often deleted in mesothelioma. The current study addresses the effect of MTAP on pemetrexed activity using a highly potent transition state inhibitor of MTAP, MT-DADMe-Immucillin A (ImmA; K(i) = 86 pmol/L) in the MTAP(+) NCI-H28 and MTAP(-) NCI-H2052 mesothelioma cell lines. Based on selective nucleoside protection, TS was found to be the primary pemetrexed target in both cell lines with GARFT inhibition requiring 20- to 30-fold higher pemetrexed concentrations. ImmA had no effect on pemetrexed activity but, when thymidine was added, the pemetrexed IC(50) decreased by a factor of approximately 3 in MTAP(+) H28 cells with no effect in MTAP(-) H2052 cells. Conversely, the transfection of MTAP into H2052 cells increased the pemetrexed IC(50) by nearly 3-fold but only in the presence of thymidine; this was reversed by ImmA. An MTAP-specific short interfering RNA produced a 2-fold decrease in pemetrexed IC(50) in MTAP(+) HeLa cells in the presence of thymidine. These data indicate that suppression of constitutive MTAP has no effect on pemetrexed activity when the primary target is TS. There is a modest salutary effect when the pemetrexed target is GARFT alone.

The inverse relationship between reduced folate carrier function and Pemetrexed activity in a human colon cancer cell line

Pemetrexed, a new generation antifolate recently approved for the treatment of mesothelioma and non-small cell lung cancer, is an excellent substrate for the reduced folate carrier (RFC). To explore the carrier's effect on pemetrexed activity, RFC was inactivated in HCT-15 colon cancer cells by mutagenesis and PT632 selective pressure. A clone (PT1) was obtained with a glycine to arginine substitution at amino acid 401, resulting in the loss of RFC function. PT1 cells were resistant to PT632 (178-fold), methotrexate (4-fold), and ZD1694 (Tomudex, raltitrexed; 20-fold), but were 3-fold collaterally sensitive to pemetrexed when grown in 25 nmol/L of 5-formyltetrahydrofolate. PT1 cells transfected with wild-type RFC had antifolate sensitivities comparable to that of wild-type HCT-15 cells, indicating that the RFC mutation was the sole basis for resistance. Folate pools were contracted in PT1 cells by 32% or 60%, as measured by radiolabeling intracellular folates or by an enzyme binding assay, respectively. This was reflected in marked (6.5-fold) collateral sensitivity to trimetrexate. The initial uptake of pemetrexed in PT1 cells was markedly reduced ( approximately 85%) but intracellular pemetrexed levels increased to approximately 60% and approximately 70% to that of wild-type cells after 2 hours and 6 days, respectively. There was increased pemetrexed inhibition of glycinamide ribonucleotide transformylase and, to a lesser extent, thymidylate synthase in PT1 cells growing in 5-formyltetrahydrofolate based on nucleoside protection analyses. Hence, loss of RFC function leads to collateral sensitivity to pemetrexed in HCT-15 cells, likely due to cellular folate pool contraction resulting in partial preservation of pemetrexed polyglutamylation and increased target enzyme inhibition. micro

Protection of Nucleosides for Oligonucleotide Synthesis

Current Protocols in Nucleic Acid ChemistryVolume 23, Issue 1 p. 2.0.1-2.0.3 Introduction Protection of Nucleosides for Oligonucleotide Synthesis Serge L. Beaucage, Serge L. BeaucageSearch for more papers by this author Serge L. Beaucage, Serge L. BeaucageSearch for more papers by this author First published: 15 September 2007 https://doi.org/10.1002/0471142700.nc0200s23Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. View the latest in Nucleic Acid Chemistry RelatedInformation

Protection of Nucleosides for Oligonucleotide Synthesis

Protection of Nucleosides for Oligonucleotide Synthesis