Characteristic And PTPs Inhibitory Activity Of α-Aminophosphatase N-derivatives And Their Copper(Ⅱ)Complexes

Protein tyrosine phosphatases (PTPs) play an important role in catalyzing protein tyrosine dephosphorylation and modulating several cellular signal transduction pathways. The dephosphorylate phosphotyrosine can regulate many cellular process, such as cellular growth, cellular differentiation, cellular metabolism, cellular migration, cellular cycle, cellular apoptosis, cell-cell communications, ion channels, gene transcription, immune response and so on. It is reported that the abnormal of PTPs activity are linked to various disease, for example, type Ⅱ diabetes mellitus (DM), obesity, cancer, neurological disorder, infections disease and angiocardiopathy. Consequently, PTPs have emerged as promising targets for therapeutic.In this paper, we designed and synthesized a series of a-aminophosphatase N-derivatives and their copper-complexes as novel, potent and selective inhibitors of protein tyrosine phosphatases, and further discussed the interaction mechanisms. The main results are listed as follow.1. Dessinged and synthesized of twenty-one a-aminophosphatase N-derivatives with N side chain having the flexible chain, the rigid, and nature L-amino acid and one cyclization product. Their compositions and structures are established by EA, UV, CD, FT-IR,1H-NMR,13C-NMR,31P-NMR and ESI-MS. Compounds L1-L4&L13(Diethyl(4-chlorophenylamino)(2-hydroxyphenyl)methylphosphonate,(L1); Diethyl (4-bromophenylamino)(2-hydroxyphenyl)methylphosphonate,(L2); Diethyl(4-nitrophenyl-amino)(2-hydroxyphenyl)methylphosphonate,(L3); Diethyl(2-chlorophenylamino)(2-hydroxyphenyl)methylphosphonate,(L4)&N-ethyl-2,3-dihydro-2-hydro-2-oxo-4,5-benzo-1,2-oxaphosphol-3-amine,(L13)) are confirmed by X-ray crystallography. The study of PTPs inhibition shows compounds L1-L5, L17, L20((2-Hydroxy-phenyl)(4-hydroxyphenylamino)methylphosphonic acid,(L5); Sodium2-((2-hydroxy-phenyl)(phosphono))methylamino)-3-hydroxy-propanoate,(L17); Sodium2-((2-hydroxy-phenyl)(phosphono))methylamino)-3-(1H-imidazol-5-yl)-propanoatetrihydrate,(L20)) are moderate competitive inhibitors with some selectivity. Compound L5is a potent and selective inhibitor of PTP1B and TCPTP with the lowest IC50value about6.64μM against PTPIB,2-fold and25-fold stronger than against TCPTP and PTP-MEG2while it doesn’t inhibit SHP-1and SHP-2. The binding constant of L5to PTP1B is2.23×105M-1with a binding ratio of approximates1:1. Cell viability and apoptosis assays indicated that L5is cell permeable with lower cytotoxicity. The results suggest a-aminophosphatase N-derivatives are possibly effective and selective inhibitors of PTPs.2. Five phosphono-containing quinquedentate ligands were employed to synthesize binuclear copper complexes,[Cu2(DPMP)2](1),[Cu2(DEMP)2](2),[Cu2(DHMP)2](3),[Cu2(D-5-Cl-PMP)2](4),[Cu2(D-5-Br-PMP)2](5), which were characterized by elemental analysis, IR, X-ray single crystal diffraction analysis, electrospray ionization mass spectra(ESI-MS), electron paramagnetic resonance(EPR), magnetisum, thermogravimetric (TG) analysis. Complexes1and2crystallized in triclinic system with the space grouppp1. Four of five-coordinated positions were filled with two N(N1,N2) and two phosphonate oxygen atoms (O1,O5,), the fifth coordinated position was occupied by021(i1-x,1-y,1-z) atom from another ligand, acting as a bridge withμ1,3-O,O phosphonate fragment linker connecting both Cu cations.The speciation of Cu-H2DPMP system in aqueous solution was investigated by pH potentiometric titrations, the results suggest the species [Cu2(DPMP)2] and [Cu?(DPMP)2H-1] are the predominant species in the physiological pH range. The five binuclear copper complexes exhibited potent and almost same inhibitory effects against protein tyrosine phosphatase1B (PTP1B) and T-cell protein tyrosine phosphatase (TCPTP) with IC50of0.16-0.24μM, about10-fold stronger inhibition than against Src homology phosphatase1(SHP-1),30-fold against Src homology phosphatase2(SHP-2) and more than100-fold against megakaryocyte protein-tyrosine phosphatase2(PTP-MEG2). Fluorescence titrations revealed that complex1bonds to the five PTPs with a molar ratio of1:1and binding constants of1.62x106,3.09×106,1.95x105,2.24x105,1.55×104M-1for PTP1B, TCPTP, SHP-1, SHP-2and PTP-MEG2respectively, consistent with the inhibitory abilities from IC5o and Ki. The three copper complexes of them could inhibit phosphatase activity of cell extracts from C6rat glioma cells as well.Farthermore, we investigated the effects of O2and GSH on the inhibition of PTP1B by complex1to probe whether the inhibition involves in reactive oxygen species produced by the Fenton reaction. The result shew that the inhibition may not involve in ROS but may be related to the oxidation state of copper.The results suggested the structures of copper complexes influence selectivity over different PTPs. In addition, the magnetic behaviors between two copper(Ⅱ) was antiferromagnetic interactions.3. The reaction of CuCl2·2H2O with EMMP(L11), EEMP(L12), EHMP(L23), EDPA(L13) lead to five new tetra-nuclear copper (Ⅱ) a-aminophosphonate cage cluster with a Double4Ring(D4R) core,(Na4[Cu4(HMPA)4]·16H2O(6·16H2O),[Na4Cu4(HE-PA)4]xH2O(7·xH2O;7a x=19,7b x=16), Na2[Cu4(HEPA)4Cu(H2O)4]·6H2O(8·6H2O), Na2[Cu4(HHPPA)4]·8H2O (9·8H2O)) and their compositions and structures are established by EA, FT-IR, second-order nonlinear optical properties(NLO), circular dichroism, magnetisum, TG analysis, electrospray ionization mass spectra (ESI-MS) and electron paramagnetic resonance(EPR). X-ray analysis shows that complexes6-9were bridged by four a-aminophosphonate acid, formed a tetraanion, and the Cu1and Cu3, Cu2and Cu4were bridged through-PO3group. In complexes6-9, the centric Cu was pentacoordinate with a distortion square pyramida. The complexes exhibited potent and almost same inhibitory effects against protein tyrosine phosphatase1B (PTP1B), T-cell protein tyrosine phosphatase (TCPTP) and SH2domain-containing protein tyrosine phosphatase1(SHP-1) with IC50at submicro molar level, about10-fold stronger inhibition than against SH2domain-containing protein tyrosine phosphatase2(SHP-2), but do not inactivate megakaryocyte protein-tyrosinephosphatase (PTP-MEG2) at100μM. At last, the complexes could inhibit phosphatase activity of cell extracts from C6rat glioma cells.In addition, the complexes exhibit second harmonic generation (SHG), anti-ferromagnet and the positive Cotton Effect (CE) by relative analysis.