Development of Novel Synthetic Oxytocin Analogues with Potential Cytostatic Activity

Cphas Muvudji Mwanasaka1, Vassiliki Magafa1, Fotini Lamari1, Zoi Diamantopoulou2, Fotini Bazoti3 Panagiotis Katsoris 2 and Paul Cordopatis1
1

Department of Pharmacy, University of Patras, GR-26500, Patras, Hellas

2Department

of Biology, University of Patras, GR-26500, Patras, Hellas

GAIA Research Center, The Goulandris Natural History Museum, GR-14562 Athens, Hellas

Introduction
Oxytocin (OT) is a cyclic nonapeptide hormone [Cys1-Tyr2-Ile3-Gln4-Asn5Cys6-Pro7-Leu8-Gly9-NH2] [fig.1] that incorporate a disulfide bridge between Cys1 and Cys6. It is produced by the hypothalamus and stored in the posterior pituitary gland [1] .Traditionally, Oxytocin (OT) is known to play a crucial role in many reproductive and behavioral functions[2, 3]. In recent OH years an additional new biological role of OT has been identified in neoplastic pathology. Through the activation of a specific G-coupled H2C oxtytocin receptor (OTR), OT may act as a growth regulator in various O H O CH3 tumors. In vitro, OT inhibits proliferation of neoplastic cells of either epithelial H2 C CHN C NH 2 H3C C CHHN (mammary, endometrial, prostatic), nervous, or bone origin, all expressing CH CH [4] . However, in neoplastic cells derived from two additional OT target OTR CH2 O C tissues, trophoblast and endothelium, OT was found to promote cell S HN S proliferation. Similarly, the proliferation of small cell lung carcinomas has H2N C H2 CH H2N CC CH2 been reported to be stimulated by OT in vitro [5]. The signal transduction H2 C O C O CH O HN CH2 pathways of OT can be different. The effect of OT on growth inhibition or CHC N C O O H growth enhancement may depend on the membrane localization of the OTR HN H2N CH2 N C itself. The OT inhibitory effect could apparently be mediated by the activation C O C O O of the cAMP-PKA pathway, a nonconventional OT signaling pathway.[6]. CH CH3 HN H2C CH Based on these data and thorough structure-activity studies from our CH3 laboratory[7], nine bicyclic oxytocin analogues (Table 1) have been Fig.1: Oxytocin molecule developed with expectation that the bicyclic structure will contribute to increased chemical stability and lipophilicity. Furthermore, two of them, the Cyclo (4-9) [Mpa1, D-Nal(1)2 , Lys4 , Thi7]oxytocin, [Mpa1 , D-Nal(1)2 , Lys4 , Thi7]oxytocin, as well as OT and Atosiban have been tested in vitro for the inhibition of proliferation on human prostate cancer cell line PC3

Results
The total yields of the synthesis of crude peptides was ranged from 75% to 80%, while the purity of the peptides after the cyclizations and purification was approximately 20-25%. The bicyclic analogues cyclo (4-8) appear significant low solubility in water, while the analogues cyclo (4-9) are absolutely insoluble. In Graph 1 the results from in vitro biological experiments of the analogue Cyclo (4-9) [Mpa1 - D-Nal(1)2 - Lys4 - Thi7] (CM04) for the inhibition of proliferation on human prostate cancer cell line PC3 compared to the relative results of THOT3, OT and Atosiban are shown. The monocyclic analogue [Mpa1 - D-Nal(1)2 - Lys4 - Thi7]oxytocin (THOT3) appears dosedependent inhibitory effect , as like Atosiban. However Atosiban appears decreased dose-dependent inhibitory effect compared to THOT3. OT shows decreased inhibitory effect compared to TOH3 and Atosiban.The bicyclic cyclo(4-9) [Mpa1 - D-Nal(1)2,Lys4 ,Thi7]oxytocin appears low and not dose-dependent inhibitory effect.

110

% Change relative to control

100

0 10 50 100

*** *** ***

90

*** *** *** ***

*** *** ***

***

80

***

70 10 0
THOT3 OT2 ATOSIBAN CMO4

Materials and Methods The new analogues were synthesized by Fmoc solid phase methodology utilizing Rink Amide MBHA and Chlorotrityl chloride (CTC) resins as solid supports and diisopropyl-carbodiimide / 1-hydroxy-benzotriazol (DIC / HOBt) as coupling reagents [8]. The cyclization Mpa1-Cys6 was performed in DMSO/H2O (10% v/v) for 24-48h, while the cyclization AA4-AA8 or AA4 - AA9 has been achieved using TBTU, DIEA, HOBT in DMF/DCM (3/7 v/v) as reagents. All the analogues were purified by gel filtration chromatography on Sephadex G-15 using 30% acetic acid as eluent and by semi preparative HPLC on reversed-phase support C-18 with linear gradient from 10 to 90% acetonitrile (0.1% TFA) for 30 min at a flow rate of 1.5 ml/min and UV detection at 220 and 254 nm . The identification of the analogues was performed by Liquid chromatography- mass spectrometry (LC-MS).The proliferation of prostate cancer cell line PC3 was evaluated by Crystal Violet method.
Mpa
HS CH2

Graph 1: inhibitory effect of New Oxytocin Analogues on the proliferation of PC3

Conclusions We have designed and synthesized 9 novel Oxytocin analogues that the main characteristic of those synthetic
analogues is that except of the S-S bond one more cyclization has been performed Cyclo (4-9) and Cyclo (4-8) . To the best of our knowledge is the first time that such kind of Oxytocin analogues have been designed, synthesized and tested for this biological target. The role of Oxyttocin and Oxytocin analogues in regulating neoplastic cell proliferation, as well as the wide distribution of OTR in neoplastic tissues can open new perspectives on the biological role of the OT-OTR system in the control of cancer growth.

Mpa -D-Nal -Ile -Lys -Asn -Cys -Thi -Leu -Gly -OH
DMSO 10%/H2O
O O HN O HN N H NH O O NH2 S S O NH NH O N NH H O O S HO

D-Tyr(Et)
H2N CH H2C O C OH

D-Nal(1)
O C OH H2N CH H2C

Aib
C H2N C CH3 CH3 HO O

Thi
O H2N CHC OH CH2 S

Tic
COOH NH

H2C C O OH

OCH 2CH3

Table 1: Developed new OT analogues and their physicochemical properties New OT Analogues HPLC tr Rf(x)

THOT3
CM01 Cyclo (4-9) [Mpa1, D-Tyr(Et)2, Lys4, Thi7] O 18,6 19,3 18.3 19. 0 17,6 18,2 18,6 18,0 18,4 0.68 0.70 0.68 0.73 0.60 0.63 0.66 0.70 0.59 CM02 Cyclo (4-9) [Mpa1, D-Tyr(Et)2 , Lys4 , D-Tic7]

References
1. Poulain, D.A., Wakerley, J.B. Neuroscience. 1982; 7: 773 2. Merlin, P. Baill Clin Obstet Gynecol. 1993; 7: 577-600 3. Soloff, M.S., Wieder, M.H. Can. J. Biochem. Cell Biol. 1983; 61: 631 4. Cassoni, P.; Marrocco, T.; Deaglio, S.; Sapino, A.; Bussolati, G. Annals of Oncology Vol.12, Sup.1, Feb.2001 , pp. 37-39(3) 5. Christel Pequeuxa, Christophe Bretonb, Marie-Therese Hagelsteina, Vincent Geenena, Legrosa j.lungcan.2005.05.027 Jean-Jacques

H2N TBTU HOBt DIEA DCM / DMF S O H N

CM03 Cyclo (4-9) [Mpa1, D-Tyr(Et)2, Lys4, D-Thi7] CM04 Cyclo (4-9) [Mpa1, D-Nal(1)2, Lys4, Thi7] CM05 Cyclo (4-8) [Mpa1, D-Tyr(Et)2, Lys4, Thi7, Glu8] CM06 Cyclo (4-8) [Mpa1, D-Tyr(Et)2, Glu4, Lys8, Aib9]
O NH O NH NH O

O HN O

S S O H2N HN O N O H O HN

CM07 Cyclo (4-8) [Mpa1, D-Nal(1)2, Glu4, Lys8, Aib9 ] CM08 Cyclo (4-8) [Mpa1, D-Nal(1)2, Lys4, Thi7, Glu8] CM09 Cyclo (4-8) [Mpa1, D-Tyr(Et)2, Lys4, Tic7, Glu8 ] X :

HN

6. Francesca Guzzi, Deborah Zanchetta, Paola Cassoni, Valeria Guzzi, Maura Francolini, Marco Parenti and Bice Chini Nature Oncogene (2002) 7. Fragiadaki M, Magafa V, Borovikova L, Slaninova J, Cordopatis P. Eur. J. Med. Chem 2007 8. Louis A. Carpino and Ayman El-Faham Tetrahedron 55 (1999) 6813-6830

TLC system (butan-1-ol/water/acetic acid/pyridine (4/1/1/2, v/v)

CM04
Fig.2: cyclization process of synthesized OT analogues (CM04)

(1)

(2)

Fig 3: Representative HPLC chromatogram of monocyclic (THOT3 ) and bicyclic (CM04 ) OT analogues(1) and the corresponding MS spectra of CM04 analogue (2)