TG-LL
Cellule normale et cellule cancéreuse
SYNTHÈSE D’ANALOGUES D- ET L-TYROSINE-CHLORAMBUCIL ACTIFS SUR LES LIGNÉES CELLULAIRES CANCÉREUSES DU SEIN
Caroline Descôteaux, Valérie Leblanc, Kevin Brasseur, Atul Gupta, Éric Asselin,
Gervais Bérubé*
Le contenu de ce chapitre a fait l’objet d’une publication en anglais dans la revue Bioorganic & Medicinal Chemistry Letters. La référence de cet article est la suivante: Bioorganic & Medicinal Chemistry Letters (2010) 20: 7388-7392. DOllO.1016/j.bmc1.201O.1O.039
Contribution des auteurs
La majeure partie des travaux présentés fut réalisée par Caroline Descôteaux, sous la supervision des Drs Bérubé et Asselin. La synthèse de l’ensemble des molécules de même que les travaux préliminaires de modélisation moléculaire ont été effectués par celle-ci. L’évaluation biologique fut également réalisée par Caroline Descôteaux et Kevi~ Brasseur, avec le soutien de Valérie Leblanc. Première auteure de cette publication, Caroline Descôteaux a rédigé, soumis, corrigé le manuscrit et répondu aux interrogations et suggestions des examinateurs avec l’appui et les conseils d’Atul Gupta et du Dr Bérubé .
Résumé
Une série d’analogues D- et L-tyrosine-chlorambucil a été synthétisé comme agents anticancéreux pour le traitement du cancer du sein. Suite à des modifications de la D- et de la L-tyrosine, les nouveaux composés ont été synthétisés avec de bons rendements.
L’évaluation biologique anticancéreuse des composés nouvellement synthétisés fut réalisée sur différentes lignées cellulaires cancéreuses du sein, hormono-dépendantes et hormono-indépendantes (RE+ et RE-). Les nouveaux analogues présentaient une activité anticancéreuse significative lorsque comparée au chlorambucil. Une étude de la relation structure-activité a permis de démontrer l’influence de la longueur d~ l’espaceur ainsi que l’influence de la stéréochimie de l’entité tyrosine. Un fait intéressant, les dérivés D-et L-tyrosinol-chlorambucil porteurs d’un espaceur constitué de 10 atomes de carbone sont plus sélectives pour les cellules cancéreuses du sein hormono-dépendantes MCF-7
(RE+).
Premier article scientifique
Synthesis of D- and L-tyrosine-chlorambucil analogs active against
breast cancer cell tines
Caroline Descôteaux, Valérie Leblanc, Kevin Brasseur, Atul Gupta, Éric Asselin,
Gervais Bérubé*
Groupe de Recherche en Oncologie et Endocrinologie Moléculaires,
Département de Chimie-Biologie, Université du Québec à Trois-Rivières, c.P. 500,
Trois-Rivières, Québec, Canada, G9A 5H7
* Corresponding author:
1-819-376-5011 extension 3353
1-819-376-5084
[email protected]
Abstract
A series of D- and L-tyrosine-chlorambucil analogs was synthesized as anticancer drugs for chemotherapy of breast cancer. The novel compounds were synthesized in good yields through efficient modifications of D- and L-tyrosine. The newly synthesized compounds were evaluated for their anticancer efficacy in different hormone-dependent and hormone-independent (ER+ and ER-) breast cancer ceH lines. The novel analogs showed significant in vitro anticancer activity when compared to chlorambucil. Structure-activity relationship (SAR) reveals both, the influence of the length of the spacer chain and the stereochemistry of the tyrosine moiety. Interestingly, the D- and L-tyrosinol-chlorambucil derivatives with 10 carbon atoms spacer are selective towards MCF-7 (ER+) breast cancer cellline.
Over the years much research has been done on breast cancer. It has been established that tumours may differ according to the protein they express.l Breast cancer is no more considered as a single homogeneous disease and treatment may differ from a patient to another. An extensive field of investigation is now to target drugs to specific molecules within the tumours and this is done by using molecular-targeted cancer therapy.
The estrogen receptor alpha (ERa) has already been used as a target to deliver a cytotoxic moiety to breast cancer cells?,3,4 ERa is a cytosolic prote in which become overexpressed in several cancerous cell types. Estradiol (E2) (1), the femalè sex hormone, binds to its receptor and then activates transcription factors and triggers proliferation (Fig. 1). Recently, our group has synthesized several E2-linked-drugs in order to carry an anticancer agent, known for its efficacy, to cells expressing the ERa. 5,6,7 These molecules have shown promising in vitro biological activity and in vivo studies of one ofthese revealed selectivity for hormone-dependent breast cancer.8
Also, non-steroidal cytotoxic hybrid molecules have already been synthesized in attempt to target the ERa.4 The aim of such design was to destroy cancerous cells without activating the ERa. Conjugates comprising a chemotherapeutic agent and an amino acid (or a derivative thereof) have also shown promising in vivo results.9 Furthermore, chlorambucil conjugates with lipidic amino acids were developed as anticancer agent. 10 This particular system was used as a tool to improve the delivery and cytotoxicity of the parent drug.
Natural amino acids have been widely used in production ofpharmaceuticals and food. 11 L-tyrosine (2-amino-3-(4-hydroxyphenyl)-propanoic acid) (2) is a natural amine acid, which plays a crucial role in human development (Fig. 1). It is the precursor for the synthesis of thyroid hormones and sorne neurotransmitters such as dopamine and noradrenaline. 12 Recently, it has been discovered that L-tyrosine and sorne of its metal complex derivatives • show antibacterial activity against Bacillus subtilis, Serratia, Pseudomonas aeruginosa and Escherichia coli. 13 It also acts as an antifungal agent.
Chlorambucil (CHL) (3) is a bis-alkylating agent which belongs to the nitrogen mustard family (Fig. 1). It is mainly used to treat chronic lymphocytic leukemia and malignant lymphomas. 14 It is also useful for the treatment of solid tumors such as advanced ovarian and breast carcinomas. 15 Like other type of anticancer treatment, administration of CHL causes non negligible toxic side effects including bone marrow suppression, anaemia and reduced immune system function. 16, 17
In our search for anticancer agents with better therapeutic index and selectivity towards breast cancer, we have designed and synthesized a new class of tyrosine-linked nitrogen mustard hybrids (4, 5) (Fig. 1). Many unusual molecular structures can bind to the ERa. 18 Thus, we hypothesized that tyrosine could be used as a building block to target the estrogen receptor as its structure could mimic, albeit slightly, the steroid backbone of 17~-estradiol (1) (Fig. 2). Thus, the phenolic group of tyrosine is used as a potential bioisosteric function imitating the phenol group of estradiol. Moreover, molecular modeling displays the possible interactions of the novel hybrids with the estrogen receptor alpha (ongoing study). This encouraged us to perform this exploratory work. Besides, the cytotoxic activity of chlorambucil and the known antibacterial activity of tyrosine could together 1) generate powerful compounds interacting with the ERa, 2) being more specific to female cancer ceUs, and 3) more potent than each of the independent components.
For this purpose, tyrosine was used as the starting material to which chlorambucil was added directly (4) or via a spacer (5) to construct several tyrosine-chlorambucil hybrids. Chlorambucil was linked to the tyrosine framework at the a-amine function . This position seems suitable as it should not affect functional groups possibly involved in the receptor recognition. The fmal compounds are either tyrosine methyl ester or tyrosinol analogs. In each case, both, natural (L-tyrosine) and non-natural (D-tyrosine) amine acids were used.
This study presents the design, synthesis and biological activities of these new tyrosine-chlorambucil conjugates (4, 5). The hybrids were made using two distinct synthetic methodologies (linear and convergent synthesis) which will be described and compared in terms of their efficiency.
With the linear synthesis (Scheme 1), tyrosine was initially transformed into the corresponding methyl ester hydrochloride salt (6) with thionyl chloride in methyl alcohol (MeOH) at reflux in 96% yield. Condensation of compound 6 with chlorambucil in presence of I-hydroxybenzotriazole (HOBt), dicyc1ohexy1carbodiimide (DCC) and triethylamine (Et3N) in N ,Ndimethylformamide (DMF) at room temperature (22°C) yielded compound 4a in 81 % yield. Borohydride reduction of compound 4a with excess of lithium borohydride (LiBH4) in dry diethyl ether (Et20) at .room temperature (22°C) gave the desired tyrosinol compounds 4b in 78% yield. Chlorambucil was also linked via a 5 or a 10 carbon atoms spacer. The amine acid spacer was first protected using 2-(t-butoxycarbonyloxyimino)-2-phenylacetonitrile (BOC-ON) in 1,4-dioxane, water and Et3N at room temperature (22°C) yielding N-BOC-amino acid (7) in 97% yield. Then, the corresponding N-Boc protected amino acid-alkyl chain (7) was linked to tyrosine methyl ester (6) via an amide function. Activation and condensation were performed using DCC and HOBt in DMF linking 7 to 6 to give compound 8 in 96% yield. Deprotection was achieved with treatment of the {Boc-compound (8) with trifluoroacetic acid in dichlorom~thane (DCM). The resulting oil was then coupled to chlorambucil (3) using DCC and HOBt in DMF. The tyrosine methyl ester chlorambucil derivatives 5a were obtained in 60% yield. The pure compounds were then reduced in the presence of LiB~ in EhO and provide the corresponding tyrosinol derivatives 5b in 57% yield.
With the convergent synthesis (Scheme 2), chlorambucil was first linked to the amino acid-spacer. The acid function of the chlorambucil was activated with isobutylchloroformate (CIC02iBu) and then coupled to the amino acid function using hexamethyldisilazane (HMDS), trimethylsilyl chloride (TMSCI), and a catalytic amount of sulfuric acid. 19 The resulting amide (9) was isolated in 98% yield. The amino acid-CHL derivative (9) was then coupled, in presence of DCC and HOBt, to the tyrosine methyl ester, obtained as described above with the linear synthesis. The tyrosine methyl ester CHL analogs (Sa) were obtained in 70% yield. The final chemical step, reduction with LiB~ in EhO, provides the desired tyrosinol derivatives in 57% yield. AH the new compounds were fuHy characterized by their respective IR, I H NMR and 13C NMR spectroscopy and by HRMS .20
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Table des matières
AVANT-PROPOS
REMERCIEMENTS
RESUME
LISTE DES FIGURES
LISTE DES ABREVI.A TIONS
CHAPITRE 1 INTRODUCTION
1.1 Épidémiologie du cancer
1.2 Histopathologie du cancer
1.2.1 Cellule normale et cellule cancéreuse
1.2.2 Cancérogénèse
1.3 Cancer du sein
1.4 Traitements
1.4.1 Chirurgie
1.4.2 Radiothérapie
1.4.3 Hormonothérapie
1.4.4 Chimiothérapie
1.5 Agents alkylants
1.5.1 Chlorambucil
1:5.2 Cisplatine
1.6 Effets secondaires
1.7 Ciblage thérapeutique
1.8 Les estrogènes
1.8.1 Synthèse des estrogènes
1.8.2 Effets physiologiques des estrogènes
1.8.3 Estradiol et cancer du sein
1.9 Le récepteur estrogénique
1.9.1 Structure
1.9.2 Isoformes alpha et bêta du récepteur aux estrogènes
1.10 Estradiol et récepteur aux estrogènes : mode d’action
1.11 Activité agoniste et antagoniste
1.12 Ligands des récepteurs aux estrogènes
1.13 Les SERMs : ligands du récepteur estrogénique
1.13.1 Les classes de SERMs
1.14 Propriétés structurales des ligands
1.15 Études antérieures
1.15.1 Encapsulation de médicaments
1.15.2 Molécules cytotoxiques conjuguées
1.15.3 Ciblage des récepteurs nucléaires
1.15.4 Molécules hybrides estradiol-cytotoxiques
1.15.5 Molécules estradiol-Pt(II) : évaluation biologique
1.15.6 Molécules hybrides antiestrogéniques cytotoxiques
1.16 Les acides aminés
1.16.1 La tyrosine
1.17 Présentation du projet de recherche
1.17.1 Hypothèses de recherche
1.17.2 Objectifs de recherche
CHAPITRE II SYNTHÈSE D’ANALOGUES D- ET L-TYROSINE-CHLORAMBUCIL ACTIFS SUR LES LIGNÉES CELLULAIRES CANCÉREUSES DU SEIN
2.1 Contribution des auteurs
2.2 Résumé
2.3 Premier article scientifique
Abstract
Acknowledgements
References and notes
CHAPITRE III ÉTUDE RSA DE RÉGIOISOMÈRES HYBRIDES TYROSINECHLORAMBUCIL; S~THÈSE ET ÉVALUATION BIOLOGIQUE SUR LES CELLULES CANCEREUSES DU SEIN
3.1 Contribution des auteurs
3.2 Résumé
3.3 Deuxième article scientifique
Abstract
Introduction
Materials and methods
Chemistry
Biology
In vitro cytotoxic activity.
Molecular modelling
Results and discussion
Synthesis oftyrosine-chlorambucil hybrid regioisomers
In vitro biological activity
Docking
Conclusion
Acknowledgments
References
CHAPITRE IV DESIGN DE NOUVELLES MOLÉCULES HYBRIDES TYROSINEMOUTARDE AZOTÉE ACTIVES SUR LES LIGNÉES CELLULAIRES CANCÉREUSES DE L’UTÉRUS, DE VOV AIRE ET DU SEIN
4.1 Contribution des auteurs
4.2 Résumé
4.3 Troisième article scientifique
Abstract
Introduction
Experimental
Chemistry
Preparation of the hydroxyphenyl-L-para-tyrosinamidechlorambucil
molecules with the linear methodology
Preparation of the hydroxyphenyl-L-para-tyrosinamidechlorambucil
molecules with the convergent methodology.
Biology
In vitro cytotoxic activity
Results and discussion
Synthe sis of ortho-, meta- and para-hydroxyphenyltyrosinamide-
chlorambucil hybrid molecules
In vitro cytotoxic activity
Conclusion
Acknowledgments
References
CHAPITRE V EXPLORATION DE LA SYNTHÈSE DE MOLÉCULES L-TYROSINEPLATINE( II) COMME AGENTS ANTICANCÉREUX POTENTIELS
5.1 Contribution des auteurs
5.2 Résumé
5.3 Quatrième article scientifique.
Abstract
Introduction
Results and discussion
Synthesis ofL-tyrosine methyl ester platinum(II) hybrids
In vitro cytotoxic activity
Conclusion
Experimental section
Chemistry
First synthetic method (Scheme 1)
Second synthetic method (Scheme 2)
Third synthetic method (Scheme 3)
Biology
In vitro cytotoxic activity
Acknowledgements
References
CHAPITRE VI DISCUSSION GÉNÉRALE
CHAPITRE VII CONCLUSION
RÉFÉRENCES BIBLIOGRAPHIQUES
ANNEXE
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