An amino acid ester derivative of luciferin (valoluc) was synthesized to mimic the transport and activation of valacyclovir. This molecule was characterized in vitro for specificity and enzymatic constants, and then assayed in two different, physiologically-relevant conditions. It was demonstrated that valoluc activation is sensitive to the same cellular factors as valacyclovir and thus has the potential to elucidate the dynamics of amino acid ester prodrug therapies in a functional, high-throughput manner.

INTRODUÇÃO: A doença de Chagas e a leishmaniose são doenças tropicais supernegligenciadas, que afetam regiões de extrema pobreza. Os fármacos disponíveis para estas duas doenças são escassos, de eficácia limitada, de alta toxicidade e suscitam casos de resistência. OBJETIVO: Considerando-se a necessidade de desenvolvimento de novos agentes antichagásicos e leishmanicidas, a importância da latenciação no aprimoramento de fármacos/compostos bioativos e a versatilidade de transportadores dendriméricos, o objetivo deste trabalho foi a síntese de pró-fármacos dendriméricos de primeira geração de 3-hidroxiflavona, composto que apresenta potencial atividade tripanomicida e leishmanicida. Desta forma, pretendeu-se obter liberação controlada, melhora da permeabilidade, toxicidade reduzida e aumento da eficácia deste agente bioativo. MATERIAL E MÉTODOS: Para a obtenção desses dendrímeros empregaram-se as abordagens divergente e convergente, compostas por várias etapas de síntese com reações de proteção, desproteção e acoplamentos. RESULTADOS E DISCUSSÃO: A abordagem convergente apresentou problemas sintéticos, devido à instabilidade dos derivados contendo 3-hidroxiflavona nas diferentes condições reacionais e de purificação testadas. No entanto, há indícios da síntese dos pró-fármacos dendriméricos de 3-hidroxiflavona, mas esses compostos apresentam-se impuros. Devido a essa instabilidade e a dificuldade de purificação na abordagem convergente, optou-se pela síntese divergente, no qual o composto bioativo é acoplado na etapa final. Os estudos sintéticos mostraram a obtenção dos intermediários puros formados pelos focos centrais propano- e hexano-diamina acoplados ao ácido málico protegido. CONCLUSÃO: Há indicativos da obtenção de pró-fármacos dendriméricos de 3-hidroxiflavona, ainda que impuros. As maiores dificuldades encontradas foram a purificação e a estabilidade dos compostos obtidos. / INTRODUCTION: Chagas\' disease and leishmaniasis are super neglected tropical diseases that affect primarily areas of extreme poverty. The drugs available for these diseases are scarce and of limited effectiveness, toxic and rouse resistance. OBJECTIVE: Considering that the development of new antichagasic and leishmanicide agents are urgently needed, the importance of prodrug design to the improvement of drugs and bioactive compounds and the versatility of dendrimers as drug carriers, the objective of this work was the synthesis of dendrimer prodrug of 3-hydroxyflavone, which shows potential antichagasic and leishmanicide activities. Thus, we intended to obtain controlled release, improvement of the permeability, reduction of the toxicity and increase of efficacy of this bioactive agent. MATERIAL AND METHODS: Convergent and divergent approaches have been used to synthesize those compounds. Synthetic steps consist of protection, deprotection and coupling reactions. RESULTS AND DISCUSSION: The convergent approach presented problems due to the instability of the 3-hydroxyflavone derivatives, in different reaction and purification conditions. However, there is evidence of the synthesis of dendrimer prodrugs, though still impure. Due to instability and purification difficulty of intermediate, we performed the divergent synthesis. We obtained the pure intermediates composed by cores propanediamine and hexanediamine coupled to the protected malic acid as spacer group. CONCLUSION: Synthetic studies suggested the synthesis of dendrimer prodrugs, although impure. The greatest difficulties were the purification and the instability of compounds.

INTRODUÇÃO: A doença de Chagas e a leishmaniose são doenças tropicais supernegligenciadas, que afetam regiões de extrema pobreza. Os fármacos disponíveis para estas duas doenças são escassos, de eficácia limitada, de alta toxicidade e suscitam casos de resistência. OBJETIVO: Considerando-se a necessidade de desenvolvimento de novos agentes antichagásicos e leishmanicidas, a importância da latenciação no aprimoramento de fármacos/compostos bioativos e a versatilidade de transportadores dendriméricos, o objetivo deste trabalho foi a síntese de pró-fármacos dendriméricos de primeira geração de 3-hidroxiflavona, composto que apresenta potencial atividade tripanomicida e leishmanicida. Desta forma, pretendeu-se obter liberação controlada, melhora da permeabilidade, toxicidade reduzida e aumento da eficácia deste agente bioativo. MATERIAL E MÉTODOS: Para a obtenção desses dendrímeros empregaram-se as abordagens divergente e convergente, compostas por várias etapas de síntese com reações de proteção, desproteção e acoplamentos. RESULTADOS E DISCUSSÃO: A abordagem convergente apresentou problemas sintéticos, devido à instabilidade dos derivados contendo 3-hidroxiflavona nas diferentes condições reacionais e de purificação testadas. No entanto, há indícios da síntese dos pró-fármacos dendriméricos de 3-hidroxiflavona, mas esses compostos apresentam-se impuros. Devido a essa instabilidade e a dificuldade de purificação na abordagem convergente, optou-se pela síntese divergente, no qual o composto bioativo é acoplado na etapa final. Os estudos sintéticos mostraram a obtenção dos intermediários puros formados pelos focos centrais propano- e hexano-diamina acoplados ao ácido málico protegido. CONCLUSÃO: Há indicativos da obtenção de pró-fármacos dendriméricos de 3-hidroxiflavona, ainda que impuros. As maiores dificuldades encontradas foram a purificação e a estabilidade dos compostos obtidos. / INTRODUCTION: Chagas\' disease and leishmaniasis are super neglected tropical diseases that affect primarily areas of extreme poverty. The drugs available for these diseases are scarce and of limited effectiveness, toxic and rouse resistance. OBJECTIVE: Considering that the development of new antichagasic and leishmanicide agents are urgently needed, the importance of prodrug design to the improvement of drugs and bioactive compounds and the versatility of dendrimers as drug carriers, the objective of this work was the synthesis of dendrimer prodrug of 3-hydroxyflavone, which shows potential antichagasic and leishmanicide activities. Thus, we intended to obtain controlled release, improvement of the permeability, reduction of the toxicity and increase of efficacy of this bioactive agent. MATERIAL AND METHODS: Convergent and divergent approaches have been used to synthesize those compounds. Synthetic steps consist of protection, deprotection and coupling reactions. RESULTS AND DISCUSSION: The convergent approach presented problems due to the instability of the 3-hydroxyflavone derivatives, in different reaction and purification conditions. However, there is evidence of the synthesis of dendrimer prodrugs, though still impure. Due to instability and purification difficulty of intermediate, we performed the divergent synthesis. We obtained the pure intermediates composed by cores propanediamine and hexanediamine coupled to the protected malic acid as spacer group. CONCLUSION: Synthetic studies suggested the synthesis of dendrimer prodrugs, although impure. The greatest difficulties were the purification and the instability of compounds.

Thesis (M.A.)--Central Connecticut State University, 2004. / Thesis advisor: Kathy Martin. " ... in partial fulfillment of the requirements for the degree of Master of Arts in Biological Sciences." Includes bibliographical references (leaf 36). Also available via the World Wide Web.

Oncolytic viruses that selectively replicate in and lyse cancer cells are a promising approach for the treatment of tumours that are resistant to conventional therapies. Clinical experience has shown that oncolytic viruses are safe and well tolerated but possess modest single agent activity. One approach to improve the efficacy of oncolytic viruses is to utilise their tumour tropism to deliver genes encoding enzymes able to activate prodrugs. ONYX-411 is an oncolytic adenovirus that replicates in cells that carry dysfunctions in the retinoblastoma (pRb) pathway, a common hallmark of cancer. ONYX-411 was ‘armed’ by inserting the Escherichia coli nfsB nitroreductase (NTR) gene into the E3B region of the viral genome under the control of the endogenous E3 viral transcriptional machinery. NTR is an oxygen-insensitive nitroreductase that is capable of activating dinitrobenzamide mustard (DNBM) prodrugs to cytotoxic metabolites. The main objective of this thesis was to determine the extent and mechanism of the therapeutic interaction between ONYX-411NTR and DNBM prodrugs. A fluorogenic probe was developed to monitor NTR activity non-invasively and revealed robust, replication dependent NTR activity in ONYX-411NTR-infected neoplastic but not primary human cell lines. In vitro exposure of ONYX-411NTR-infected cells to therapeutically relevant concentrations of the DNBM prodrugs (SN 27686 or PR-104A) did not inhibit virus replication. Tumour growth delay studies of systemic ONYX-411NTR followed by prodrug demonstrated different outcomes in three models (H1299, C33A, 22Rv1). To establish predictable viral infection of tumours a pre-infection model was developed using HCT 116 xenografts. This methodology demonstrated that prodrug administration (SN 28343 or PR-104) provided significant inhibition of tumour growth without suppression of ONYX-411NTR replication. Follow-on studies using intravenous virus administration confirmed titre amplification with time (24-fold between day 3 and 13 post administration; P < 0.001) and a marked survival gain for the virus/prodrug combinations. Neither the prodrugs nor ONYX-411NTR were active as single agents. The improvement in efficacy for the combination of ONYX-411NTR and prodrug was conditional on NTR-dependent prodrug activation resulting in improved virus distribution within the tumour. PR-104 is currently in clinical development making the combination of ONYX-411NTR with PR-104 a promising strategy for cancer selective therapy. / Whole document restricted, but available by request, use the feedback form to request access.

Oncolytic viruses that selectively replicate in and lyse cancer cells are a promising approach for the treatment of tumours that are resistant to conventional therapies. Clinical experience has shown that oncolytic viruses are safe and well tolerated but possess modest single agent activity. One approach to improve the efficacy of oncolytic viruses is to utilise their tumour tropism to deliver genes encoding enzymes able to activate prodrugs. ONYX-411 is an oncolytic adenovirus that replicates in cells that carry dysfunctions in the retinoblastoma (pRb) pathway, a common hallmark of cancer. ONYX-411 was ‘armed’ by inserting the Escherichia coli nfsB nitroreductase (NTR) gene into the E3B region of the viral genome under the control of the endogenous E3 viral transcriptional machinery. NTR is an oxygen-insensitive nitroreductase that is capable of activating dinitrobenzamide mustard (DNBM) prodrugs to cytotoxic metabolites. The main objective of this thesis was to determine the extent and mechanism of the therapeutic interaction between ONYX-411NTR and DNBM prodrugs. A fluorogenic probe was developed to monitor NTR activity non-invasively and revealed robust, replication dependent NTR activity in ONYX-411NTR-infected neoplastic but not primary human cell lines. In vitro exposure of ONYX-411NTR-infected cells to therapeutically relevant concentrations of the DNBM prodrugs (SN 27686 or PR-104A) did not inhibit virus replication. Tumour growth delay studies of systemic ONYX-411NTR followed by prodrug demonstrated different outcomes in three models (H1299, C33A, 22Rv1). To establish predictable viral infection of tumours a pre-infection model was developed using HCT 116 xenografts. This methodology demonstrated that prodrug administration (SN 28343 or PR-104) provided significant inhibition of tumour growth without suppression of ONYX-411NTR replication. Follow-on studies using intravenous virus administration confirmed titre amplification with time (24-fold between day 3 and 13 post administration; P < 0.001) and a marked survival gain for the virus/prodrug combinations. Neither the prodrugs nor ONYX-411NTR were active as single agents. The improvement in efficacy for the combination of ONYX-411NTR and prodrug was conditional on NTR-dependent prodrug activation resulting in improved virus distribution within the tumour. PR-104 is currently in clinical development making the combination of ONYX-411NTR with PR-104 a promising strategy for cancer selective therapy. / Whole document restricted, but available by request, use the feedback form to request access.

Oncolytic viruses that selectively replicate in and lyse cancer cells are a promising approach for the treatment of tumours that are resistant to conventional therapies. Clinical experience has shown that oncolytic viruses are safe and well tolerated but possess modest single agent activity. One approach to improve the efficacy of oncolytic viruses is to utilise their tumour tropism to deliver genes encoding enzymes able to activate prodrugs. ONYX-411 is an oncolytic adenovirus that replicates in cells that carry dysfunctions in the retinoblastoma (pRb) pathway, a common hallmark of cancer. ONYX-411 was ‘armed’ by inserting the Escherichia coli nfsB nitroreductase (NTR) gene into the E3B region of the viral genome under the control of the endogenous E3 viral transcriptional machinery. NTR is an oxygen-insensitive nitroreductase that is capable of activating dinitrobenzamide mustard (DNBM) prodrugs to cytotoxic metabolites. The main objective of this thesis was to determine the extent and mechanism of the therapeutic interaction between ONYX-411NTR and DNBM prodrugs. A fluorogenic probe was developed to monitor NTR activity non-invasively and revealed robust, replication dependent NTR activity in ONYX-411NTR-infected neoplastic but not primary human cell lines. In vitro exposure of ONYX-411NTR-infected cells to therapeutically relevant concentrations of the DNBM prodrugs (SN 27686 or PR-104A) did not inhibit virus replication. Tumour growth delay studies of systemic ONYX-411NTR followed by prodrug demonstrated different outcomes in three models (H1299, C33A, 22Rv1). To establish predictable viral infection of tumours a pre-infection model was developed using HCT 116 xenografts. This methodology demonstrated that prodrug administration (SN 28343 or PR-104) provided significant inhibition of tumour growth without suppression of ONYX-411NTR replication. Follow-on studies using intravenous virus administration confirmed titre amplification with time (24-fold between day 3 and 13 post administration; P < 0.001) and a marked survival gain for the virus/prodrug combinations. Neither the prodrugs nor ONYX-411NTR were active as single agents. The improvement in efficacy for the combination of ONYX-411NTR and prodrug was conditional on NTR-dependent prodrug activation resulting in improved virus distribution within the tumour. PR-104 is currently in clinical development making the combination of ONYX-411NTR with PR-104 a promising strategy for cancer selective therapy. / Whole document restricted, but available by request, use the feedback form to request access.

Oncolytic viruses that selectively replicate in and lyse cancer cells are a promising approach for the treatment of tumours that are resistant to conventional therapies. Clinical experience has shown that oncolytic viruses are safe and well tolerated but possess modest single agent activity. One approach to improve the efficacy of oncolytic viruses is to utilise their tumour tropism to deliver genes encoding enzymes able to activate prodrugs. ONYX-411 is an oncolytic adenovirus that replicates in cells that carry dysfunctions in the retinoblastoma (pRb) pathway, a common hallmark of cancer. ONYX-411 was ‘armed’ by inserting the Escherichia coli nfsB nitroreductase (NTR) gene into the E3B region of the viral genome under the control of the endogenous E3 viral transcriptional machinery. NTR is an oxygen-insensitive nitroreductase that is capable of activating dinitrobenzamide mustard (DNBM) prodrugs to cytotoxic metabolites. The main objective of this thesis was to determine the extent and mechanism of the therapeutic interaction between ONYX-411NTR and DNBM prodrugs. A fluorogenic probe was developed to monitor NTR activity non-invasively and revealed robust, replication dependent NTR activity in ONYX-411NTR-infected neoplastic but not primary human cell lines. In vitro exposure of ONYX-411NTR-infected cells to therapeutically relevant concentrations of the DNBM prodrugs (SN 27686 or PR-104A) did not inhibit virus replication. Tumour growth delay studies of systemic ONYX-411NTR followed by prodrug demonstrated different outcomes in three models (H1299, C33A, 22Rv1). To establish predictable viral infection of tumours a pre-infection model was developed using HCT 116 xenografts. This methodology demonstrated that prodrug administration (SN 28343 or PR-104) provided significant inhibition of tumour growth without suppression of ONYX-411NTR replication. Follow-on studies using intravenous virus administration confirmed titre amplification with time (24-fold between day 3 and 13 post administration; P < 0.001) and a marked survival gain for the virus/prodrug combinations. Neither the prodrugs nor ONYX-411NTR were active as single agents. The improvement in efficacy for the combination of ONYX-411NTR and prodrug was conditional on NTR-dependent prodrug activation resulting in improved virus distribution within the tumour. PR-104 is currently in clinical development making the combination of ONYX-411NTR with PR-104 a promising strategy for cancer selective therapy. / Whole document restricted, but available by request, use the feedback form to request access.

Solid tumors are commonly subject to hypoxia. Hypoxic cancer cells have undesirable properties such as a high tendency to metastasize and resistance to chemotherapy and radiotherapy. Hypoxia-inducible factors (HIFs) respond to the changes in oxygen levels, orchestrating the transcription of many proteins that are vital for the survival of hypoxic cancer cells. With their parent drug SN-38 as an inhibitor of both topoisomerase 1 and HIF-1, hypoxia-activated SN-38s may have a dual inhibitory effect on hypoxic tumor cells due to hypoxia-targeting and HIF-1 inhibition. To develop hypoxia-activated prodrugs of SN-38; 2-, 3-, and 4-nitrobenzyl SN-38s have been synthesized with good yields (78%, 67% and 68%, respectively). Topoisomerase 1 inhibitory assay on 2- and 4-nitrobenzyl SN-38s and cell viability assay on 2-, 3- and 4-nitrobenzyl SN-38s have been performed. All three derivatives showed less toxicity on K562 cells, which meets the principle of prodrug design. Cyclic voltammetry results suggest that the reduction potentials of these three derivatives may be not high enough for these compounds to be activated. The manner of reduction of three nitrobenzyl SN-38s is quasi-reversible under the testing condition, not against the proposed mechanism of activation. Two new derivatives of SN-38 have been designed to elevate reduction potential and further reduce toxicity. They are to be synthesized and tested for future work. / October 2016

<p>J1 (L-melphalanyl-L-<i>p</i>-fluorophenylalanyl ethyl ester) is a dipeptide derivative of the alkylating agent melphalan with increased cytotoxicity. In this thesis the preclinical pharmacology of J1 has been characterized.</p><p>Our results show that J1 rapidly enters the cells, where melphalan is released by hydrolysis. The maximum concentration (C_max) of melphalan was detected 15 min after exposure to J1 in human cancer cell lines. In comparison, melphalan exposure resulted in a 10-fold lower C_max that was shifted to later time points. J1 induced more DNA damage and apoptosis than melphalan. The cytotoxic activity and release of melphalan from J1 were inhibited by preincubating cells with the aminopeptidase inhibitor bestatin. In accordance with these results, we showed that J1 is a substrate for aminopeptidase N (APN), which may result in increased tumor selectivity.</p><p>J1 effectively inhibited cell growth in a set of neuroblastoma cell lines.<i> </i>Athymic mice carrying neuroblastoma xenografts were treated either with equimolar doses of melphalan or J1. J1 inhibited the tumor growth more effectively than melphalan and the untreated control, and was associated with higher caspase-3 activation, fewer proliferating tumor cells and decreased mean vascular density.</p><p>J1 and melphalan showed similar activity profiles when tested in 176 primary tumor cell cultures from patients, but J1 exhibited 50- to 100-fold higher potency. The difference was greater in some diagnoses (e.g. breast cancer, NHL and AML), and was exceptionally large in some breast cancer samples with aggressive phenotypes. A combination screening of J1 and standard chemotherapeutics yielded mostly additive interactions, except for etoposide which induced synergy in all tested cell lines.</p><p>In conclusion, the melphalan prodrug J1 is effectively transported into the cells, where aminopeptidases (for example APN) catalyze the formation of melphalan. J1 shows promising preclinical potential in the diagnoses neuroblastoma and breast cancer.</p>