A proteína hnRNP K é conhecida por seu papel nos múltiplos processos que compõe a expressão gênica, incluindo funções nos estágios de splicing, transcrição e tradução, desempenhadas, principalmente, através da ligação de seus domínios KH a nucleotídeos. A ativação inadequada da hnRNP K tem relação direta com a gênese de alguns tipos de câncer, sobretudo de cabeça e pescoço, mama e colo-retal, evidenciando a mesma como um atrativo alvo molecular para o desenvolvimento de novos fármacos antineoplásicos. Com o auxílio de técnicas in silico, foram identificados dois compostos orgânicos, um derivado de benzimidazol e outro derivado de fenilbenzamida, capazes de impedir a ligação da proteína hnRNP K a oligonucleotídeos in vitro. Aliando as técnicas de docking, campos de interação e dinâmica molecular foi possível sugerir que tais compostos apresentam características estruturais que permitem a realização de interações na fenda de ligação do domínio KH3, principalmente com os resíduos R40 e R59, os quais são considerados chaves no reconhecimento molecular de nucleotídeos. Os derivados de benzimidazol e fenilbenzamida constituem novos compostos de partida na busca de novos antineoplásicos que tem como alvo a proteína hnRNP K. Do ponto de vista de metabolismo e toxicidade, o derivado de fenilbenzamida parece ser mais promissor quando se investiga uma molécula para aplicação terapêutica, uma vez que gerou poucos alertas críticos de toxicidade, ao contrario do derivado de benzimidazol, que apresenta maior potencial genotóxico. Apesar de se ligarem aos domínios KH da hnRNP K e impedir sua complexação a nucelotídeos, ensaios com culturas de células mostraram apenas tênue atividade antitumoral para tais compostos, com maior redução de viabilidade celular, ao redor de 18% a 8,4 M, exibida pelo derivado de fenilbenzamida. Seguindo o princípio do análogo ativo, simulações de triagem virtual na busca de análogos dos ligantes da hnRNP K revelaram 21 novos derivados de benzimidazol ou fenilbenzamida na base de dados EXPRESS-Pick, dos quais 5 foram testados in vitro com a proteína e 3 novos ligantes identificados. Com o intuito de otimizar tais derivados, foram sugeridos in silico substituintes (potenciais bioisósteros) para os anéis dioxopirrolidínicos dos ligantes já identificados, guiando, assim, a futura síntese de novas substâncias com potencial atividade antitumoral. Além disso, o trabalho foi complementado através da proposição de síntese de novos derivados benzoxazepínicos acoplados a purinas, os quais também tem aplicação como antineoplásicos, entretanto por mecanismos que não envolvem a hnRNP K. A grande limitação desses derivados é a presença de um grupo nitro aromático, o qual é reconhecido por sua toxicidade pronunciada. Com o intuito de otimizar tais derivados, foram sugeridos in silico potenciais bioisósteros capazes de substituir o grupo nitro e guiar a síntese e novos derivados com atividade antitumoral e toxicidade reduzida. / hnRNP K protein is known for its role in the multiple processes that compose gene expression, including functions during splicing, transcription and translation, developed, mainly, by the binding of nucleotides to KH domains. Inadequate activation of hnRNP K induces the development of some types of cancer, including head and neck, breast and colorectal. In this way, hnRNP K is an attractive molecular target for antineoplastic drug design. Using in silico strategies, we have identified two organic compounds, a benzimidazole and a phenylbenzamide derivatives, able to prevent the natural binding of nucleotides to hnRNP K in vitro. Applying docking, molecular interaction fields and molecular dynamics simulations it was possible to propose that such compounds present structural characteristics capable to support intermolecular interactions inside KH3 domain binding cleft, mainly with R40 and R59 residues, which are extremely important during molecular recognition of nucleotides by hnRNP K. The benzimidazole and phenylbenzamide derivatives identified are novel lead compounds that can guide the design of new antineoplastic drugs targeting hnRNP K. Considering metabolic and toxicity predictions, the phenylbenzamide seems to be more promising than the benzimidazole derivative as a drug, once the benzimidazole presents genotoxic potential. Although both derivatives prevent the binding of nucleotides to hnRNP K, biological assays with tongue cancer cell lines revealed only a mild antitumoral activity for such compounds. Higher level of cell viability reduction, 18% at 8.4 M, was observed for the phenylbenzamide derivative. Following the similarity principle, virtual screening simulations were made intending to find novel benzimidazole and phenylbenzamide derivatives inside EXPRESS-Pick database. The search revealed 21 compounds, 5 of which were tested in vitro with hnRNP K, where 3 of them were active. Intending to optimize benzimidazole and phenylbenzamide derivatives in order to design more potent chemical entities, we have suggested in silico substituents as potential bioisosteric groups of the dioxopyrrolidine rings of hnRNP K ligands, guiding the future synthesis of novel compounds with enhanced antitumoral activity. Moreover, complementary work proposition was performed through the synthesis of benzoxazepin-purines, which also present antitumoral activity but not through hnRNP K pathway. The major limitation of such derivatives is the presence of a nitro aromatic group, which can be very toxic. 20 potential bioisosteric groups were proposed as fragment candidates to replace the nitro one in order to design novel antitumoral derivatives with reduced toxic potential.

A proteína hnRNP K é conhecida por seu papel nos múltiplos processos que compõe a expressão gênica, incluindo funções nos estágios de splicing, transcrição e tradução, desempenhadas, principalmente, através da ligação de seus domínios KH a nucleotídeos. A ativação inadequada da hnRNP K tem relação direta com a gênese de alguns tipos de câncer, sobretudo de cabeça e pescoço, mama e colo-retal, evidenciando a mesma como um atrativo alvo molecular para o desenvolvimento de novos fármacos antineoplásicos. Com o auxílio de técnicas in silico, foram identificados dois compostos orgânicos, um derivado de benzimidazol e outro derivado de fenilbenzamida, capazes de impedir a ligação da proteína hnRNP K a oligonucleotídeos in vitro. Aliando as técnicas de docking, campos de interação e dinâmica molecular foi possível sugerir que tais compostos apresentam características estruturais que permitem a realização de interações na fenda de ligação do domínio KH3, principalmente com os resíduos R40 e R59, os quais são considerados chaves no reconhecimento molecular de nucleotídeos. Os derivados de benzimidazol e fenilbenzamida constituem novos compostos de partida na busca de novos antineoplásicos que tem como alvo a proteína hnRNP K. Do ponto de vista de metabolismo e toxicidade, o derivado de fenilbenzamida parece ser mais promissor quando se investiga uma molécula para aplicação terapêutica, uma vez que gerou poucos alertas críticos de toxicidade, ao contrario do derivado de benzimidazol, que apresenta maior potencial genotóxico. Apesar de se ligarem aos domínios KH da hnRNP K e impedir sua complexação a nucelotídeos, ensaios com culturas de células mostraram apenas tênue atividade antitumoral para tais compostos, com maior redução de viabilidade celular, ao redor de 18% a 8,4 M, exibida pelo derivado de fenilbenzamida. Seguindo o princípio do análogo ativo, simulações de triagem virtual na busca de análogos dos ligantes da hnRNP K revelaram 21 novos derivados de benzimidazol ou fenilbenzamida na base de dados EXPRESS-Pick, dos quais 5 foram testados in vitro com a proteína e 3 novos ligantes identificados. Com o intuito de otimizar tais derivados, foram sugeridos in silico substituintes (potenciais bioisósteros) para os anéis dioxopirrolidínicos dos ligantes já identificados, guiando, assim, a futura síntese de novas substâncias com potencial atividade antitumoral. Além disso, o trabalho foi complementado através da proposição de síntese de novos derivados benzoxazepínicos acoplados a purinas, os quais também tem aplicação como antineoplásicos, entretanto por mecanismos que não envolvem a hnRNP K. A grande limitação desses derivados é a presença de um grupo nitro aromático, o qual é reconhecido por sua toxicidade pronunciada. Com o intuito de otimizar tais derivados, foram sugeridos in silico potenciais bioisósteros capazes de substituir o grupo nitro e guiar a síntese e novos derivados com atividade antitumoral e toxicidade reduzida. / hnRNP K protein is known for its role in the multiple processes that compose gene expression, including functions during splicing, transcription and translation, developed, mainly, by the binding of nucleotides to KH domains. Inadequate activation of hnRNP K induces the development of some types of cancer, including head and neck, breast and colorectal. In this way, hnRNP K is an attractive molecular target for antineoplastic drug design. Using in silico strategies, we have identified two organic compounds, a benzimidazole and a phenylbenzamide derivatives, able to prevent the natural binding of nucleotides to hnRNP K in vitro. Applying docking, molecular interaction fields and molecular dynamics simulations it was possible to propose that such compounds present structural characteristics capable to support intermolecular interactions inside KH3 domain binding cleft, mainly with R40 and R59 residues, which are extremely important during molecular recognition of nucleotides by hnRNP K. The benzimidazole and phenylbenzamide derivatives identified are novel lead compounds that can guide the design of new antineoplastic drugs targeting hnRNP K. Considering metabolic and toxicity predictions, the phenylbenzamide seems to be more promising than the benzimidazole derivative as a drug, once the benzimidazole presents genotoxic potential. Although both derivatives prevent the binding of nucleotides to hnRNP K, biological assays with tongue cancer cell lines revealed only a mild antitumoral activity for such compounds. Higher level of cell viability reduction, 18% at 8.4 M, was observed for the phenylbenzamide derivative. Following the similarity principle, virtual screening simulations were made intending to find novel benzimidazole and phenylbenzamide derivatives inside EXPRESS-Pick database. The search revealed 21 compounds, 5 of which were tested in vitro with hnRNP K, where 3 of them were active. Intending to optimize benzimidazole and phenylbenzamide derivatives in order to design more potent chemical entities, we have suggested in silico substituents as potential bioisosteric groups of the dioxopyrrolidine rings of hnRNP K ligands, guiding the future synthesis of novel compounds with enhanced antitumoral activity. Moreover, complementary work proposition was performed through the synthesis of benzoxazepin-purines, which also present antitumoral activity but not through hnRNP K pathway. The major limitation of such derivatives is the presence of a nitro aromatic group, which can be very toxic. 20 potential bioisosteric groups were proposed as fragment candidates to replace the nitro one in order to design novel antitumoral derivatives with reduced toxic potential.

Εξήντα έξη χρόνια μετά την εμφάνιση της πρώτης δημοσίευσης που αφορούσε στη θεραπεία με τη χρήση μουσταρδών αζώτου και ενώσεις που ανήκουν στη θεραπευτική αυτή κατηγορία, όπως το κυκλοφωσφαμίδιο και η μελφαλάνη εξακολουθούν να αποτελούν πρώτης γραμμής χημειοθεραπευτικούς παράγοντες τόσο κατά της λευχαιμίας όσο και έναντι άλλων συμπαγών όγκων. Οι διλειτουργικοί αλκυλιωτικοί παράγοντες, κατηγορία στην οποία ανήκουν και οι μουστάρδες αζώτου, επάγουν το θάνατο των καρκινικών κυττάρων και κατά συνέπεια και την κυτταροτοξική τους δράση μέσω της δημιουργίας διαμοριακών χιαστί τύπου δεσμών μεταξύ των κλώνων του DNA. Οι φαρμακολογικές τους δράσεις σχετίζονται κυρίως με την σύνθεση του DNA των διαιρούμενων κυττάρων, προκαλώντας ουσιαστικά μια γενική αναστολή της κυτταρικής διαίρεσης. Ωστόσο, εκτός από την αναμενόμενη και επιθυμητή θεραπευτική δράση, προκαλούν τοξικές παρενέργειες, οι οποίες σχετίζονται με μεταλλαγή των κυττάρων της αρχέγονης σειράς, εμφάνιση καρκινογένεσης και τερατογένεσης. Το εύρος των μειονεκτημάτων που παρουσιάζουν και ειδικά η συστηματική τοξικότητα σε συνδυασμό με την υψηλή χημική in vivo δραστικότητα τους και την μειωμένη εκλεκτικότητα τους, είχαν ως αποτέλεσμα τον περιορισμό της χρήσης τους. Παρόλα αυτά κατέχουν εξέχουσα θέση στη θεραπευτική έναντι μιας μεγάλης ποικιλίας τύπων καρκίνου, συμπεριλαμβανομένου και των αιματολογικών νεοπλασμάτων. Στην προσπάθεια επίλυσης του προβλήματος της αυξημένης τοξικότητας και της απουσίας εκλεκτικότητας των ενώσεων της κατηγορίας αυτής, διάφορες μουστάρδες αζώτου συζεύχθηκαν μέσω εστερικού δεσμού με ποικίλους στεροειδικούς σκελετούς, στρατηγική που αποδείχθηκε ουσιαστικά ιδιαιτέρως αποτελεσματική. Αποτελέσματα μελετών τόσο της ερευνητικής μας ομάδας όσο και άλλων, υποστηρίζουν πως ο στεροειδικός σκελετός δεν μπορεί πλέον να θεωρηθεί μόνο ως απλός φορέας για την αποτελεσματική μεταφορά της αλκυλιωτικής ομάδας στις θέσεις στόχους του DNA, εφόσον μικρές δομικές τροποποιήσεις του επιφέρουν σημαντικές διαφοροποιήσεις στην αντιλευχαιμική δράση των τελικών στεροειδών εστέρων. Επιπρόσθετα, οι μελέτες αυτές έδειξαν ότι η παρουσία της –NHCO- ομάδας είτε ως ενδοκυκλικής λακταμικής είτε ως εξωκυκλικής αμιδικής στον Δ-δακτύλιο του στεροειδικού τμήματος είναι καθοριστικός παράγοντας για την εμφάνιση κυτταροτοξικής ή αντινεοπλασματικής δράσης στεροειδών εστέρων της χλωραμβουκίλης και αναλόγων αυτής. Μέχρι σήμερα έχει προκύψει ένας εκτεταμένος αριθμός στεροειδών εστέρων, από την σύζευξη απλών και τροποποιημένων στεροειδών με αρκετές διαφορετικές μουστάρδες αζώτου. Η πλειονότητα αυτών των παραγώγων έχουν υποβληθεί σε in vitro και in vivo βιολογικές δοκιμές έναντι των λευχαιμίων Ρ388 και L1210 και αρκετά από αυτά έχουν αποδειχθεί αποτελεσματικοί αντινεοπλασματικοί παράγοντες. Σε μια πρόσφατη 3D-QSAR μελέτη τριάντα οκτώ στεροειδών εστέρων τριών διαφορετικών μουσταρδών αζώτου με απλούς και τροποποιημένους στεροειδικούς σκελετούς, έγινε προσπάθεια διερεύνησης των 3D σχέσεων χημικής δομής-βιολογικής δραστικότητας, με σκοπό την εύρεση των απαραίτητων στερεοηλεκτρονικών χαρακτηριστικών των ενώσεων που συμβάλλουν στην εκδήλωση της αντιλευχαιμικής δράσης των. Η ανάλυση στηρίχθηκε στην χρήση των μεθοδολογιών CoMSIA και CoMFA. Τα δεδομένα που ελήφθησαν από τις 3D-QSAR αναλύσεις αξιοποιήθηκαν στη συνέχεια από ένα υπολογιστικό πρόγραμμα de novo σχεδιασμού και ανακάλυψης νέων βιοδραστικών μορίων, την εφαρμογή LeapFrog. Με βάση τα είκοσι βελτιωμένα στεροειδικά εστερικά παράγωγα που προτάθηκαν από το LeapFrog, επιλέχθηκαν να σχεδιαστούν και να συντεθούν οι ακόλουθες αρωματικές μουστάρδες αζώτου: α) 2-[π-Ν,Ν-δις(2-χλωροαιθυλ)αμινοφαινυλο]βουτυρικό οξύ (7), β) 2-ακεταμιδο-2-[π-Ν,Ν-δις(2-χλωροαιθυλ)αμινοφαινυλο]οξικό οξύ (30), γ) 2-ακεταμιδο-2-[ο-Ν,Ν-δις(2-χλωροαιθυλ)αμινοφαινυλο]οξικό οξύ (31) και δ) το 3-ακεταμιδο-3-[π-Ν,Ν-δις(2-χλωροαιθυλ)αμινοφαινυλο] προπανοϊκό οξύ (42), καθώς και τα εστεροποιημένα παράγωγα αυτών, με επιλεγμένους απλούς και τροποποιημένους στεροειδικούς σκελετούς. Η σύζευξη των νεων μουσταρδών με επιλεγμένες στεροειδικές αλκοόλες απέδωσε δεκαεπτά νέα εστερικά ανάλογα, η σύνθεση των οποίων είχε ως απώτερο σκοπό τη βελτίωση του θεραπευτικού δείκτη των μουσταρδών αζώτου καθώς και τον εμπλουτισμό της βιβλιοθήκης των αποτελεσμάτων που αφορούν στη σχέση χημικής δομής-βιολογικής δραστικότητας των ενώσεων της κατηγορίας αυτής. Συγκεκριμένα, για τη σύνθεση των παραπάνω μουσταρδών αζώτου, εφαρμόστηκε η κλασσική μεθοδολογία σύνθεσης. Ωστόσο, αυτή αποδείχθηκε αποδοτική μονό κατά τη σύνθεση της μουστάρδας 7, παρέχοντας το τελευταίο σε συνολική απόδοση 45%. Για τις υπόλοιπες μουστάρδες ακολουθήθηκαν εναλλακτικές πορείες σύνθεσης λόγω προβλημάτων που παρουσιάστηκαν κατά την εφαρμογή της κλασσικής μεθοδολογίας. Κρίσιμο στάδιο κατά τη σύνθεση των μουσταρδών αζώτου 30 και 31, αποτέλεσε η αρχική προστασία της ελεύθερης αμινομάδας της DL-α-φαινυλγλυκίνης (16) που χρησιμοποιήθηκε ως πρώτη ύλη της συνθετικής πορείας. Η διερεύνηση και η βελτιστοποίηση των πειραματικών συνθηκών ήταν απαραίτητη και κατά το στάδιο της χλωρίωσης των Ν,Ν-δις(2-υδροξυαιθυλ)-παραγώγων 26 και 27 για τον σχηματισμό της χαρακτηριστικής Ν,Ν-δις(2-χλωροαιθυλαμινο)-ομάδας των παραγώγων 30 και 31. Κατά τη σύνθεση του 3-ακεταμιδο-3-[π-Ν,Ν-δις(2-χλωροαιθυλ)αμινοφαινυλο]προπανοϊκού οξέος (42) ήταν απαραίτητη η βελτιστοποίηση παρασκευής των ενδιαμέσων 33 και 37. Μετά από διερευνητικές προσπάθειες κατέστη εφικτή η βελτίωση της απόδοσης σύνθεσης του παραγώγου 33 από 54% σύμφωνα με τη βιβλιογραφία σε 98%, καθώς και η σύνθεση του ακετάμιδο παραγώγου 37 τόσο σε λιγότερα συνθετικά στάδια και σε καλύτερη απόδοση σε σχέση με τα δημοσιευμένα βιβλιογραφικά δεδομένα. Σε επόμενο στάδιο της μελέτης σχεδιάσθηκε η εστεροποίηση των μουστάρδων 7, 30 και 42 με επιλεγμένους στεροειδικούς σκελετούς. Για τη σύνθεση των τελικών εστερικών στεροειδικών παραγώγων εφαρμόστηκε η μέθοδος των μικτών ανυδριτών, η οποία περιλαμβάνει την αντίδραση της αρωματικής μουστάρδας αζώτου με ελαφρά περίσσεια 2,4,6-τριχλωροβενζοϋλοχλωρίδιου παρουσία τριαιθυλαμίνης, το σχηματισμό του αντίστοιχου μικτού ανυδρίτη και στη συνέχεια in situ αντίδρασή του με τη στεροειδική αλκοόλη παρουσία 4-διμεθυλαμινοπυριδίνης. Τελικώς κατέστη εφικτή η σύνθεση δεκαεπτά τελικών στεροειδών εστέρων από τη σύζευξη των μουσταρδών αζώτου, 7 και 42 με επιλεγμένες στεροειδείς αλκοόλες. Παρά τις διερευνητικές προσπάθειες που καταβλήθησαν δεν κατέστη εφικτή η εστεροποίηση της μουστάρδας 30. Το σύνολο των τελικών στεροειδών εστέρων προωθήθηκαν σε βιολογικές μελέτες για την αποτίμηση της αντινεοπλασματικής τους δράσης. Τα διαθέσιμα αποτελέσματα αυτών των μελετών σχετίζονται με τα έντεκα στεροειδικά εστερικά παράγωγα της μουστάρδας 7 (2-PHE-BU). Τα μελετηθέντα αυτά παράγωγα επέδειξαν μειωμένη τοξικότητα και μια οριακή αντινεοπλασματική δραστικότητα έναντι της λευχαιμίας Ρ388, στην οποία είχαν μελετηθεί, συγκριτικά με την ελέυθερη αρωματική μουστάρδα (2-PHE-Bu). Ωστόσο, τα ληφθέντα βιολογικά αποτελέσματα ήταν υποδεέστερα των αντίστοιχων δεδομένων που είχαν συλλεχθεί κατά τη μελέτη του PHE και των στεροειδικών εστέρων αυτού έναντι και πάλι της λευχαιμίας Ρ388. Το γεγονός αυτό πιθανόν να οφείλεται στη μειωμένη ενζυμική υδρόλυση που υφίστανται in vivo τα παράγωγα αυτά, λόγω της στερεοχημικής παρεμπόδισης που προκαλεί η αιθυλική αλυσίδα γύρω από τον εστερικό δεσμό σύνδεσης μουστάρδας-στεροειδούς, γεγονός που συνεπάγεται μειωμένη απελευθέρωση της μουστάρδας αζώτου. Στο σημείο αυτό αξίζει να αναφερθεί πως αν και, τόσο οι νέες αρωματικές μουστάρδες αζώτου, όσο και τα στεροειδικά τους παράγωγα είχαν προταθεί ως υποψήφια παράγωγα με βελτιωμένη βιοδραστικότητα κατά την πραγματοποίηση de novo σχεδιασμού από το υπολογιστικό πρόγραμμα LeapFrog, τα αποτελέσματα των βιολογικών δοκιμών δεν επιβεβαιώσαν τις συγκεκριμένες ποτάσεις. Πιθανότατα, παράμετροι που δεν ελήφθησαν υπόψη κατά τον de novo σχεδιασμό από το LeapFrog, όπως η μεταβολική τροποποίηση των ενώσεων να εμπλέκονται και να καθορίζουν την βιοδραστικότητα του τελικών στεροειδικών εστέρων. Θεωρείται ότι μια αναθεώρηση κρίσιμων παραμέτρων όπως η χημική ποικιλομορφία και ο αριθμός των ενώσεων που εισέρχονται ως δεδομένα στις μελέτες 3D-QSAR μπορεί να αποδώσει μεγαλύτερης αξιοπίστιας μοντέλα CoMFA και CoMSIA. Τα νέα αυτά μοντέλα κατά την εισαγωγή τους σε υπολογιαστικά προγράμματα de novo σχεδιασμού μπορούν να συμβάλλουν στο σχεδιασμό νεών ενώσεων με πιθανά υπέρτερη βιοδραστικότητα. / Sixty-six years after the serendipitous discovery and the first publication referred to nitrogen mustard therapy, nitrogen mustards such as cyclophosphamide and melphalan are still front-line chemotherapeutic agents for the treatment of leukemia and various solid tumors. Several chemotherapeutics act as DNA-damaging agents resulting in cell cycle arrest and cell death of the uncontrollably proliferating cancer cells. Among them, bifunctional DNA alkylating agents, such as the nitrogen mustards, form intrestrand crosslinks (ICLs), extremely cytotoxic lesions, blocking DNA replication and transcription. However, cellular responses triggered by ICLs can cause resistance in tumor cells, limiting the efficacy of such treatment. Cytotoxicity, mutagenesis, and clastogenesis are attributed to their ability to damage DNA. Although, these drugs remain some of the most commonly prescribed chemotherapies for the treatment of various solid and hematological malignancies, particularly in combination with other classical or target therapeutics in multi-agent regimens, severe side effects on normal tissues comprise drawbacks of their use. These are attributed to their low selectivity to alkylate specific DNA bases due to their high inherent reactivity, resulting in the non-specific alkylation of other cellular nucleophilic species such as amino acid residues or low molecular weight thiols. Several approaches have been explored to reduce the toxicity and increase the therapeutic efficacy of nitrogen mustards. Among them, the generation of DNA-directed alkylating agents via the chemical linkage of nitrogen mustards with molecules of increased DNA-binding affinity and the synthesis of nitrogen mustard prodrugs led to interesting results. The chemical linkage of nitrogen mustards to carrier molecules (e.g steroids) with affinity for specific binding sites (nuclear receptors) has been used aiming at the improvement of the antineoplastic treatment. Our ongoing studies in this field have demonstrated that steroidal esteric derivatives of aromatic nitrogen mustards increase the damaging effects on specific DNA sequences and achieve better selectivity and reduced toxicity compared to nitrogen mustards themselves. Steroidal skeletons, which incorporate a –NHCO- moiety are considered more appropriate modules than the common or non-modified steroids, since their esters with aromatic nitrogen mustards, such as chlorambucil and its analogs, have been proved potent antileukemic agents. To this direction, our group has published a series of studies related with steroidal esters of aromatic nitrogen mustards as antineoplastic, especially antileukemic agents. Our efforts have succeeded in the identification of potent and promising derivatives with enhanced activity and reduced toxicity compared to the corresponding nitrogen mustards against in vitro and in vivo experimental tumors. Extensive structure-activity relationship (SAR) studies have demonstrated unique structural features of both the steroidal part and the nitrogen mustard which contribute substantially to the bioactivity of the target steroidal esters Furthermore, recent 3D QSAR/CoMFA and CoMSIA studies led to the generation of related models which indicated the influence of stereoelectronic and physicochemical parameters on the antileukemic activity of target compounds. The reliability of both models was evaluated and their predictive ability on the activity of a test set of compounds proved satisfactory. Furthermore, based on the proposed CoMFA model and using the de novo ligand design routine LeapFrog of SYBYL, a series of candidate molecules with potentially optimal bioactivity was proposed, creating new challenges in further investigation of this class of compounds. Prompted by the aforementioned results and extending our structure-activity relationship studies, we decided to investigate if the incorporation of in silico predictive nitrogen mustards on various simple and modified steroids might result to steroidal esters with improved antineoplastic activity. The selection of the nitrogen mustards which were synthesized and used in this study was based on the predictive antileukemic activity of their corresponding steroidal esters and the synthetic accessibility of the intermediate and target compounds. Thus, we designed and synthesized a new series of steroidal esters containing the aromatic nitrogen mustards 2-[4-N,N-bis(2-chloroethyl)amino-phenyl]butanoic acid (2-PHE-BU, 7), 2-acetamido-2-[p-N,N-bis(2-chloroethyl)amino-phenyl]acetic acid (30), 2-acetamido-2-[o-N,N-bis(2-chloroethyl)amino-phenyl]acetic acid (31) and 3-acetamido-3-[p-N,N-bis(2-chloroethyl)amino-phenyl]propanoic acid (42). The chemical linkage of nitrogen mustards 7 and 42 with simple and modified steroids led to the synthesis of seventeen new steroidal esters aiming at the investigation of new compounds with enhanced antineoplastic activity and higher therapeutic index. The synthesis of the aforementioned nitrogen mustards was based on a classic synthetic approach. However, this procedure proved efficient only for the synthesis of compound 7, in an overall yield of 45%. Key step for the synthesis of nitrogen mustards 30 and 31 was the protection of the free amino-group of DL--phenyglycine. Optimization of the experimental conditions were also necessary for the chlorination of N,N-bis(2-hydroxyethyl) intermediates 26 and 27, which led to the corresponding nitrogen mustards 30 and 31. During the synthesis of the 3-acetamido-3-[p-N,N-bis(2-chloroethyl)amino]propanoic acid (42), optimization of the procedure for the preparation of the intermediates 33 and 37 was also necessary. Our attempts resulted in the preparation of compound 37 in less steps and higher yield (98%) compared to that referred in the literature. The synthesis of the final steroidal esteric derivatives was based on the well established method of mixed anhydrides, which involves the reaction of the aromatic nitrogen mustard with a slight excess of 2,4,6-trichlorobenzoyl chloride in the presence of triethylamine, followed by the addition of steroidal alcohol in the presence of 4-dimethylaminopyridine. The newly synthesized alkylating steroidal esters exhibited reduced toxicity and slightly improved antileukemic activity against P388 leukemia bearing mice compared to the free nitrogen mustard 2-PHE-BU. Nevertheless, they did not prove superior to already synthesized, structurally related steroidal esters of PHE indicating that the enzymatic hydrolysis and the liberation of the nitrogen mustard in vivo is possibly disfavored due to the steric hindrance of the ethyl group around the formed ester bond. Nevertheless, the biological results obtained in the present study clearly indicate that except of the important stereoelectronic requirements which are considered and incorporated as data in LeapFrog routine, other parameters are possibly implicated and determine the bioactivity of the target steroidal esters. We expect that a careful revision of the implicated parameters such as the chemical diversity and the number of the tested compounds will allows us the generation of new CoMFA and CoMSIA models which subsequently will contribute to higher predicted reliability of the in silico design by software packages such as LeapFrog.

The thesis specializes in the computational description of pharmaceutically important compounds. A substantial number of pharmaceutical drugs are small molecules that are bound to an active site of an enzyme by the "lock (binding site) and key (drug)" model through non-covalent interactions. The association of enzymes with drugs cause an increase or decrease in the activity of enzymes. The main topic is focused on the computational elucidation of the structural basis for the interactions of the purine-like compounds with the enzyme cyclin- dependent kinase 2 that belongs to the protein-kinase enzyme family. These enzymes play an important role in the cell cycle regulation; their increased activity significantly contributes to the loss of control over cell proliferation, which is one of the primary causes of cancer cell formation. The study describes the binding motifs of roscovitine, which shows an inhibitory effect on the function of cyclin-dependent kinases, and its analogues containing bioisosteric central heterocycles in the complex with cyclin-dependent kinase 2. The binding affinity between the cyclin-dependent kinase 2 enzyme and the inhibitors was quantified as calculated binding scores and evaluated in relation to the conformation of the optimized structures. The hybrid model combining the...