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Mitochondria play essential roles in numerous cellular processes, including oxidative phosphorylation and apoptotic initiation. As a result, organellar dysfunction has been implicated in several pathologies such as cancer, diabetes and neurodegenerative diseases. The opportunity to deliver compounds to probe or treat these conditions would be highly beneficial but accessing this organelle is challenging. Prior work investigated the physicochemical properties required for mitochondrial targeting and yielded mitochondria-penetrating peptides (MPPs). MPPs possess hydrophobic and cationic character and exhibit efficient cellular uptake and mitochondrial localization. In this proof-of-principle study, MPPs were harnessed to re-route an anti-leukemia agent, chlorambucil (Cbl), from the nucleus to mitochondria. This DNA alkylating agent was selected for its rapid kinetics and facile conjugation to an MPP. In addition, because mitochondria possess their own genome, the target of this drug would also be present in the organelle. Conjugation of an MPP to Cbl (mt-Cbl) confirmed that the drug was re-routed to the mitochondria and an increase in potency was observed in several cell lines and patient samples. This gain in activity was due to the increased accessibility of the mitochondrial genome, its lack of introns and its limited repair capacity. However, despite this enhanced toxicity, a therapeutic window continued to be maintained due to the elevated mitochondrial membrane potential in cancer cells. The re-routing of Cbl also resulted in evasion of several drug resistance mechanisms. Damage directly within the organelle was sufficient to initiate apoptosis even in cell lines with disabled apoptotic triggering. In addition, mitochondrial sequestration protected mt-Cbl from drug inactivation mechanisms. Lastly, mt-Cbl inhibited Pgp efflux by unexpectedly interacting with the pumps and inhibiting activity for a short period of time. The anti-cancer activity of mt-Cbl was also assessed in vivo in xenograft models of leukemia. The conjugate was stable in mouse plasma and displayed an improved pharmacokinetic profile. In addition, mt-Cbl successfully delayed tumor growth in two xenograft models and continued to alkylate mitochondrial DNA in vivo. These studies demonstrate that MPPs can be harnessed to re-route drugs to this organelle. Mitochondrial re-targeting could be a novel method of re-purposing FDA-approved drugs to enhance activity and evade resistance.

Mitochondria play essential roles in numerous cellular processes, including oxidative phosphorylation and apoptotic initiation. As a result, organellar dysfunction has been implicated in several pathologies such as cancer, diabetes and neurodegenerative diseases. The opportunity to deliver compounds to probe or treat these conditions would be highly beneficial but accessing this organelle is challenging. Prior work investigated the physicochemical properties required for mitochondrial targeting and yielded mitochondria-penetrating peptides (MPPs). MPPs possess hydrophobic and cationic character and exhibit efficient cellular uptake and mitochondrial localization. In this proof-of-principle study, MPPs were harnessed to re-route an anti-leukemia agent, chlorambucil (Cbl), from the nucleus to mitochondria. This DNA alkylating agent was selected for its rapid kinetics and facile conjugation to an MPP. In addition, because mitochondria possess their own genome, the target of this drug would also be present in the organelle. Conjugation of an MPP to Cbl (mt-Cbl) confirmed that the drug was re-routed to the mitochondria and an increase in potency was observed in several cell lines and patient samples. This gain in activity was due to the increased accessibility of the mitochondrial genome, its lack of introns and its limited repair capacity. However, despite this enhanced toxicity, a therapeutic window continued to be maintained due to the elevated mitochondrial membrane potential in cancer cells. The re-routing of Cbl also resulted in evasion of several drug resistance mechanisms. Damage directly within the organelle was sufficient to initiate apoptosis even in cell lines with disabled apoptotic triggering. In addition, mitochondrial sequestration protected mt-Cbl from drug inactivation mechanisms. Lastly, mt-Cbl inhibited Pgp efflux by unexpectedly interacting with the pumps and inhibiting activity for a short period of time. The anti-cancer activity of mt-Cbl was also assessed in vivo in xenograft models of leukemia. The conjugate was stable in mouse plasma and displayed an improved pharmacokinetic profile. In addition, mt-Cbl successfully delayed tumor growth in two xenograft models and continued to alkylate mitochondrial DNA in vivo. These studies demonstrate that MPPs can be harnessed to re-route drugs to this organelle. Mitochondrial re-targeting could be a novel method of re-purposing FDA-approved drugs to enhance activity and evade resistance.

The main goals of this research were to synthesize nitrogen oxide releasing diazeniumdiolates and their prodrugs and to evaluate their pharmacological effects. The different projects and their results are described below. i. Comparison of HNO and NO donating properties of cyclic amine diazeniumdiolates Diazeniumdiolates are an attractive class of donor compounds as they can be tuned to release NO or both NO and HNO depending upon the amine backbone. Isopropylamine (IPA/NO) and cyclohexylamine (CHA/NO) diazeniumdiolates are currently the only examples of primary amine based diazeniumdiolates. A series of structurally related cyclic amine based diazeniumdiolates were synthesized and characterized. An acetoxymethyl derivative was also synthesized to facilitate cellular uptake and to achieve higher HNO levels in cells. ii. Nitrogen oxide releasing diazeiumdiolate based adducts of N-des-methyl-tamoxifen Nitrogen oxide (NO/HNO) donating diazeniumdiolate adducts of N-desmethyltamoxifen (a key metabolite of the breast cancer drug tamoxifen) were synthesized. DEA/NO-AcOM, an NO donor was also synthesized to monitor the effect of NO on breast cancer cell survival. Derivatives of N-desmethyltamoxifen were found to be effective towards estrogen receptor positive (ER+) cells only. DEA/NO-AcOM was found to be cytotoxic towards estrogen-dependent and independent cell lines, in combination with tamoxifen, or by itself. iii. Synthesis and characterization of nitrogen oxide adducts with non-steroidal anti-inflammatory drugs (NSAIDs) Our group has shown HNO releasing diazeniumdiolate derivatized aspirin to be comparably effective in preventing gastric ulceration to NO-releasing diazeniumdiolate based aspirin analogues. Series of such NSAID adducts were further extended by synthesizing such derivatives of indomethacin and niflumic acid. NO/HNO releasing analogues of aspirin and indomethacin were cytotoxic towards two different breast cancer cell lines, irrespective of estrogen dependence.iv. Chlorambucil analogue of PABA/NOChlorambucil, an alkylating agent is used in leukemia treatment. Tumor cells resistant to alkylating agents often have increased glutathione levels and increased activity of glutathione-S-transferase (GST). PABA/NO is an NO donor with a promising anticancer profile. The chlorambucil analogue of PABA/NO was synthesized to utilize GST for releasing NO and to potentially overcome cellular resistance.

Εστιάζοντας στα αποτελέσματα προσφάτων μελετών, οι οποίες είχαν ως θέμα τη διερεύνηση της σχέσης μεταξύ της χημικής δομής και της αντιλευχαιμικής δράσης, προκύπτει ότι το στεροειδικό τμήμα των ενώσεων που περιέχουν αλκυλιωτικούς παράγοντες, δεν αποτελεί μόνο τον βιολογικό φορέα των αλκυλιωτικών παραγόντων. Ο σχεδιασμός και η μελέτη παρόμοιων ενώσεων, οι οποίες έφεραν στη θέση 7 του στεροειδικού σκελετού μία αλλυλική κετόνη, παρουσίασαν μία σημαντικά αυξημένη αντιλευχαιμική δραστικότητα συγκριτικά με παράγωγα που περιείχαν στο μόριό τους έναν απλό στεροειδικό σκελετό. Προκειμένου να διερευνηθεί κατά πόσο η βελτίωση της αντιλευχαιμικής οφειλόταν στην εισαγωγή της 7-κετόνης ή του Δ5-7-κετο στεροειδικού συστήματος, αναγάγαμε το διπλό δεσμό στη θέση 5 του στεροειδούς. Τα 5α-7-κετο στεροειδικά παράγωγα που προέκυψαν κατά την αναγωγή του διπλού δεσμού χρησιμοποιήθηκαν αρχικά για τη σύνθεση των στεροειδικών εστέρων της χλωραμβουκίλης και του φαινυλοξικού οξέος και ακολούθως μελετήθηκε η in vivo αντιλευχαιμική τους δράση έναντι της λευχαιμίας Ρ388. Τέλος, η αναγωγή του διπλού δεσμού αποδείχθηκε να έχει αρνητική επίδραση στην αντιλευχαιμική δράση, αφού η συγκριτική μελέτη με τα πρωτότυπα μόρια έδειξε πως οι πολύ δραστικοί Δ5-7-κετο στεροειδικοί εστέρες με την αναγωγή του διπλού δεσμού μετατράπηκαν σε ενώσεις με αμελητέα αντιλευχαιμική δράση. / Recent structure-antileukemic activity studies showed that the steroidal part of complex molecules containing DNA alkylators does not play only the role of the “biological carrier”. New such compounds designed to possess an allylic 7-ketone showed enhanced antileukemic potency compared with derivatives with a simple steroidal skeleton. In order to investigate whether the enhancement of the antileukemic potency is attributed to the introduction of the 7-ketone or to the Δ5-7-keto conjugated steroidal system we decided to reduce the Δ5 double bond. The 5α-7-keto-steroidal skeletons synthesized were tethered to chlorambucil and phenyl acetic acid’s nitrogen mustard and studied against leukemia P338 in vivo. The reduction of the double bond had a negative impact on the antileukemic potency since the comparative study of the novel derivatives showed that a series of very potent Δ5-7-keto-steroidal esters were converted by this modification to compounds with marginally accepted activity.

Our lab recently demonstrated that retargeting an alkylating agent, chlorambucil (Cbl), to the mitochondrion is a viable strategy to restore drug activity and overcome drug resistance in cancer. The mechanism of action of the mitochondria-targeted chlorambucil (mt-Cbl) was studied using a cervical carcinoma model. It was discovered that mt-Cbl bound to mitochondrial DNA and various mitochondrial proteins. A ρ° model revealed that the toxicity of mt-Cbl is largely dependent on its protein targets. Damage induced by mt-Cbl was found to result in the activation of several modes of caspase-independent cell death including necrosis. In contrast, Cbl was found to only activate caspase-dependent cell death that is highly sensitive to caspase inhibition. These results illustrate that the ability of mt-Cbl to activate various orthogonal cell death pathways is what allows mt-Cbl to bypass several drug resistance mechanisms, thus making mitochondrial retargeting a lucrative strategy for future anticancer drug development.

Our lab recently demonstrated that retargeting an alkylating agent, chlorambucil (Cbl), to the mitochondrion is a viable strategy to restore drug activity and overcome drug resistance in cancer. The mechanism of action of the mitochondria-targeted chlorambucil (mt-Cbl) was studied using a cervical carcinoma model. It was discovered that mt-Cbl bound to mitochondrial DNA and various mitochondrial proteins. A ρ° model revealed that the toxicity of mt-Cbl is largely dependent on its protein targets. Damage induced by mt-Cbl was found to result in the activation of several modes of caspase-independent cell death including necrosis. In contrast, Cbl was found to only activate caspase-dependent cell death that is highly sensitive to caspase inhibition. These results illustrate that the ability of mt-Cbl to activate various orthogonal cell death pathways is what allows mt-Cbl to bypass several drug resistance mechanisms, thus making mitochondrial retargeting a lucrative strategy for future anticancer drug development.

Το φυσικό προϊόν αρτεμισινίνη και τα παράγωγά της αποτελούν σήμερα φάρμακα επιλογής για την αντιμετώπιση της ελονοσίας ενώ πολλά απ’αυτά παρουσιάζουν και ιδιαίτερα σημαντική αντικαρκινική δράση. Στο πλαίσιο της παρούσας διπλωματικής εργασίας έγινε χημική τροποίηση του μορίου της αρτεμισίνης, προκειμένου να συντεθούν διμερή συζεύγματά της με πολυαμίνες (πουτρεσκίνη, σπερμιδίνη, σπερμίνη). Αρχικά, η αρτεμισινίνη τροποποιήθηκε κατάλληλα έτσι ώστε να φέρει συνδέτη με δεσμό C-O (10-oξo) ή C-C (10-καρβο) στη θέση-10 και N-C με αλλαγή του ετεροατόμου στην θέση-11, προκειμένου στη συνέχεια να προσδεθεί στις πολυαμίνες μέσω δεσμού ουρεθάνης. Για το λόγο αυτό, συντέθηκαν ενεργοποιημένα ανάλογα της αρτεμισινίνης, τα οποία μετά από αντίδραση με κατάλληλα προστατευμένες πολυαμίνες, οδηγούν στο σχηματισμό διμερών πολυαμινικών συζευγμάτων της αρτεμισινίνης. Επιπλέον, για προκαταρκτικές βιολογικές μελέτες, συντέθηκε ένα ασύμμετρο πολυαμινικό σύζευγμα της αρτεμισινίνης με το αντικαρκινικό φάρμακο χλωραμβουκίλη, χρησιμοποιώντας το 10-oξo ενεργοποιημένο ανάλογο και την πουτρεσκίνη. / The natural product Artemisinin and its derivatives are currently the drugs of choice for the treatment of malaria, which some of them showing important anticancer activity. In the context of the present dissertation, three chemical modifications of the molecule of artemisinin were accomplished towards the synthesis of several Artemisinin dimer conjugates with polyamines (putrescine, spermidine, spermine). Initially, artemisinin was modified at position 10 and 11, in order to synthesize analogues bearing suitable linkers, through C-O (10-oxo), C-C (10-carbo) and C-N (11-aza, by replacement of O with N in the ring A of Artemisinin) bonds, able to form carbamate bonds with amino groups of polyamines. For this purpose, the corresponding activated intermediates were synthesized, which upon reaction with suitably protected polyamines afforded the Artemisinin symmetric conjugates. In addition, for the sake of preliminary biological evaluation a new asymmetric conjugate consisted of an 10-oxo Artemisinin and a chlorambucil moiety, using putrescine as a polyamine-type linker, was synthesized.

Το φυσικό προϊόν αρτεμισινίνη και τα παράγωγά της αποτελούν σήμερα φάρμακα επιλογής για την αντιμετώπιση της ελονοσίας ενώ πολλά απ’αυτά παρουσιάζουν και ιδιαίτερα σημαντική αντικαρκινική δράση. Στο πλαίσιο της παρούσας διπλωματικής εργασίας έγινε χημική τροποποίηση του μορίου της αρτεμισινίνης, προκειμένου να συντεθούν διμερή συζεύγματά της με πολυαμίνες (πουτρεσκίνη, σπερμιδίνη, σπερμίνη). Αρχικά, η αρτεμισινίνη τροποποιήθηκε κατάλληλα έτσι ώστε, να φέρει συνδέτη με δεσμό C-O (10-oxo) στη θέση-10, προκειμένου στη συνέχεια να προσδεθεί στις πολυαμίνες μέσω δεσμού ουρεθάνης. Για το λόγο αυτό, συντέθηκε το ενεργοποιημένο ανάλογο(10-oxo , το οποίο μετά από αντίδραση με τις κατάλληλα προστατευμένες πολυαμίνες, έδωσαν τα διμερή συζεύγματα της αρτεμισινίνης 66-68. Επιπλέον, για προκαταρκτικές μελέτες βιολογικής δράσης, συντέθηκε ένα ασύμμετρο σύζευγμα της αρτεμισινίνης με το αντικαρκινικό χλωραμβουκίλη (69), χρησιμοποιώντας το 10-oxo ενεργοποιημένο ανάλογο και την πουτρεσκίνη ως πολυαμίνη. Όλα τα παραπάνω συζεύγματα, βρίσκονται στο στάδιο της αποτίμησης της βιολογικής τους δράσης, από συνεργαζόμενο εργαστήριο έναντι καρκινικών σειρών HL60. / The natural product Artemisinin and its derivatives are currently the drugs of choice for the treatment of malaria, with a large number of them showing important anticancer activity as well. In the context of the present dissertation, one chemical modification of artemisinin was accomplished towards the synthesis of several Artemisinin dimer conjugates with polyamines (putrescine, spermidine, spermine). Initially, artemisinin was modified at the 10- position, in order to synthesize analogue bearing suitable linkers, through C-O (10-oxo) bond, able to form urethane bonds with amino groups of polyamines. For this purpose, the activated intermediate(10-oxo) was synthesized, which upon reaction with suitably protected polyamines afforded the Artemisinin symmetric conjugates 66-68. In addition, for the sake of preliminary biological evaluation a new asymmetric conjugate (69) consisted of an 10-oxo Artemisinin and a chlorambucil moiety, using putrescine as a polyamine-type linker, was synthesized. All above conjugates are now under biological evaluation as anticancer agents, against HL60 cancer cells, by a collaborating research group. Further studies are underway, in order to evaluate the biological activity of the aforementioned analogues as antimalarials.

This diploma thesis is focused on the determination of chloramphenicol by differential pulse voltammetry (DPV), DC voltammetry (DCV), cyclic voltammetry (CV) and adsorptive stripping voltammetry (AdSV) on a meniscus modified silver solid amalgame electrode (m- AgSAE). For the determination of chloramphenicol the optimum conditions were found and under these conditions concentration dependences were measured and then limits of quantification were determined. The influence of pH of BR buffer was tested. For determination of chloramphenicol by DCV pH 7 was chosen as an optimum background and pH 8 was chosen as an optimmum for DPV determination. Under these conditions linear dependences were obtained in the concentration range of 1·10-6 - 1·10-4 mol·l-1 . The limit of detection of chloramphenicol by DCV was 2.3·10-6 mol·l-1 . The limit of detection of chloramphenicol by DPV was 2.1·10-6 mol·l-1 in distilled water, 2.9·10-6 mol·l-1 in river water and 4.2·10-6 mol·l-1 in drinking water. Electrochemical behavior of chloramphenicol was studied by cyclic voltammetry in BR buffer with pH 2, 6, 8, 12 and then mechanism of reduction of chloramphenicol was propose based on available literature. Chloramphenicol was determined in drug Spersadex by DPV in BR buffer with pH 8. The optimal conditions for determining...