Synthesis of cyclic peptide homologs of glutathione as potential antitumor agents

Two cyclic tripeptide homologs, cyclo(Glu[Cys-β-Ala-]-OH) 8a, and cyclo(Glu[Cys-Gaba-]-OH) 8b, were synthesized by the pentafluorophenyl ester method in solution (1-3). These cyclic peptides are cyclo homologs of glutathione and are designed as potential antitumor agents. The ¹H- and ¹³C-n.m.r. spectral parameters of cyclo(Glu[Cys(Bzl)-β-Ala-]-OH) 7a were measured in DMSO-d₆ and a possible conformation has been proposed. The cyclic peptide 8a showed low cytotoxic activities against three human tumor cell lines: KB, HeLa, and Colo 205.     

Activity of RA-700, a cyclic hexapeptide from Rubiae Radix,in the human tumor clonogenic assay

In vitro phase II study of a new cyclic hexapeptide anticancer agent, RA-700 was studied on the human tumor clonogenic assay. From the results of the study using the human tumor cell line of lung cancer (PC-6), RA-700 appears to possess time-dependent antitumor activity. Therefore, against the 148 human specimens of various malignancies, the chemosensitivity of RA-700 was tested at the concentrations of 10 g/ml, 1 g/ml and 0.1 g/ml in continuous exposure schedule for 2 weeks by using the human tumor clonogenic assay. If the criteria for in vitro sensitivity was based on 70% inhibition of colony formation, out of 148 specimens 59 specimens (40%) were evaluable and the chemosensitivity rate of RA-700 were 67% (4/6), 22% (2/9), 17% (3/18) and 10% (1/10) for ovarian cancer, non-small cell lung cancer, breast cancer and colorectal cancer, respectively. An overall chemosensitivity rate against 13 different histologic types of cancers was 22% (13/59) ( 70% inhibition of colony formation) and 39% (23/59) ( 50% inhibition of colony formation). RA-700 showed almost same chemosensitivity compared to that of five standard anticancer drugs (adriamycin, mitomycin C, cisplatin, vinblastine and 5-FU), but the spectrum of RA-700 activity appears to be different from that of the standard drugs. Furthermore, the antitumor activity of RA-700 had no relationship with prior chemotherapy. These results indicated that RA-700 is a candidate for phase I study.     

Structure-based rational design of peptide hydroxamic acid inhibitors to target tumor necrosis factor-α converting enzyme as potential therapeutics for hepatitis

The human tumor necrosis factor-α converting enzyme (TACE) has recently been raised as a new and promising therapeutic target of hepatitis and other inflammatory diseases. Here, we reported a successful application of the solved crystal structure of TACE complex with a peptide-like ligand INN for rational design of novel peptide hydroxamic acid inhibitors with high potency and selectivity to target and inhibit TACE. First, the intermolecular interactions between TACE catalytic domain and INN were characterized through an integrated bioinformatics approach, with which the key substructures of INN that dominate ligand binding were identified. Subsequently, the INN molecular structure was simplified to a chemical sketch of peptide hydroxamic acid compound, which can be regarded as a linear tripeptide capped by a N-terminal carboxybenzyl group (chemically protective group) and a C-terminal hydroxamate moiety (coordinated to the Zn²⁺ at TACE active site). Based on the sketch, a virtual combinatorial library containing 180 peptide hydroxamic acids was generated, from which seven samples were identified as promising candidates by using a knowledge-based protein–peptide affinity predictor and were then tested in vitro with a standard TACE activity assay protocol. Consequently, three designed peptide hydroxamic acids, i.e. Cbz-Pro-Ile-Gln-hydroxamic acid, Cbz-Leu-Ile-Val-hydroxamic acid and Cbz-Phe-Val-Met-hydroxamic acid, exhibited moderate or high inhibitory activity against TACE, with inhibition constants K_i of 36?±?5, 510?±?46 and 320?±?26?nM, respectively. We also examined the structural basis and non-bonded profile of TACE interaction with a designed peptide hydroxamic acid inhibitor, and found that the inhibitor ligand is tightly buried in the active pocket of TACE, forming a number of hydrogen bonds, hydrophobic forces and van der Waals contacts at the interaction interface, conferring both stability and specificity for TACE–inhibitor complex architecture.     

Synthesis and preliminary biological evaluation of a 99mTc‐chlorambucil derivative as a potential tumor imaging agent

Technetium‐99m‐based radiopharmaceuticals have been used widely as diagnostic agents in the nuclear medicine. Chlorambucil (CLB) as one typical alkylating drug exhibits excellent inhibition effects against many human malignancies. To develop and explore a novel potential imaging agent for early diagnosis of tumors, tricarbonyl technetium‐99m and rhenium complexes on the basis of the tridentate ligand dipicolylamine (DPA) bound to the chlorambucil pharmacophore were designed and synthesized: ^99mTc‐DPA‐CLB ( 3 ) and Re‐DPA‐CLB ( 4 ). The high performance liquid chromatography analyses showed that the retention time of 3 and 4 was 13.5 and 13.6 minutes, respectively. Radiolabeling efficiency of the ^99mTc‐DPA‐CLB tracer was 97%, and the radiochemical purity was larger than 95% after 6 hours stored in phosphate buffered saline or human serum as observed by thin layer chromatography and high performance liquid chromatography. Biodistribution studies in a mouse model of breast cancer showed ^99mTc‐DPA‐CLB exhibited a favorable tumor affinity. The radiotracer cleared quickly in the first hour via hepatobiliary and renal routes of excretion, resulted in a very low background at 4 hours post injection (p.i.). It had moderate uptake ratios of tumor to blood and tumor to muscle. These results suggested ^99mTc‐DPA‐CLB might be a promising SPECT imaging agent for tumor diagnosis.     

An in vitro assessment of the antineoplastic potential of 2H-1,3-Oxazine-2,6(3H)-dione (3-oxauracil), a novel pyrimidine

The pyrimidine (uracil) analogue 3-oxauracil (OU) previously had been shown to completely inhibit the growth of E. coli B and decrease by 96% the replication of herpes simplex virus type 2 when present in the culture fluid at a concentration of 10² µM. Limited in vivo studies in mice demonstrated antiviral effects without significant toxicity when given i.p. daily for two weeks at a concentration of 3.23 mg/kg. However, the antineoplastic properties of OU were unknown. We assessed the ability of OU to inhibit the proliferation of various human tumor cell lines (3 pancreatic, 1 colon, 1 neuroendocrine, and 1 lung) in an in vitro radiometric (Bactec) system. In the pancreatic lines (RWP-2, MiaPaCa-2, and PANC-1), the colon line (HT-29), the neuroendocrine line (COLO 320DM), and the lung cancer cell line (SK-MES-1), OU at a concentration of 10³ µM, produced a dramatic decrease in percent cell survival. When compared with cytotoxic drugs of choice for these tumor cells (gemcitabine, 5-fluorouracil, and adriamycin, respectively) a significantly higher concentration of OU was required usually to achieve comparable results with two exceptions. These were the HT-29 and the COLO 320DM cell lines. These results indicate OU has significant (p < 0.05) cytotoxic activity against pancreatic, colon, neuroendocrine, and nonsmall cell lung cancer lines, when compared to untreated control cultures. Additional in vivo testing of this potential antineoplastic agent is warranted.     

Efficient methods for the synthesis of 5‐(4‐[18F]fluorophenyl)‐10,15,20‐tris(3‐methoxyphenyl)porphyrin as a potential imaging agent for tumor

F‐18‐labeled porphyrins, the potential tracing and detecting agents for tumor have been synthesized and characterized by two convenient routes: one is a mixed aldehyde condensation, which involves acid‐catalyzed condensation of pyrrole, m‐anisaldehyde and 4‐[¹⁸F]fluorobenzaldehyde. The other is the acid‐catalyzed condensation of tetrapyrrane with 4‐[¹⁸F]fluorobenzaldehyde. The synthetic methodologies including solvents, reaction concentrations and catalysts are optimized for radiolabeled porphyrins. The methods also provide the desired product in reasonable radiochemical yield (20–26%) compared with those of cold chemical synthesis (1–3%) and with high radiochemical purity (>95%). The methods described here would be effective and convenient ways to produce radiolabeled porphyrin. Copyright © 2005 John Wiley & Sons, Ltd.     

Synthesis and activity evaluation of 3′‐floxuridinyl 4‐[3‐(3, 5‐di‐t‐butyl‐4‐methoxyphenyl)‐3‐oxo‐propenyl]benzoate: in vitro and in vivo as a potential dual‐acting antitumor prodrug

BOBA (4‐[3‐(3, 5‐di‐tert‐butyl‐4‐methoxyphenyl)‐3‐oxo‐propenyl]benzoic acid), a substituted chalcone derivative, exhibits an excellent inducing differentiation on neoplastic cellular differentiation. FUDR (5‐fluoro‐2′‐deoxyuridine, floxuridine) inhibits DNA biosynthesis and has been used extensively to treat various cancers. In our efforts to find a new dual‐action antitumor prodrug, 3′‐floxuridinyl 4‐[3‐(3, 5‐di‐t‐butyl‐4‐methoxyphenyl)‐3‐oxo‐propenyl] benzoate (3′‐O‐BOBA‐ FUDR) was synthesized, and its antiproliferative activity in vitro and antitumor efficacy in vivo were evaluated. Compared with FUDR, the antiproliferative activity of 3′‐O‐BOBA‐FUDR was improved by 3–7‐fold. In rat hepatocellular carcinoma xenografts, 3′‐O‐BOBA‐FUDR‐treated rats had smaller tumors than were found in controls. In addition, the expression of Bcl‐2 protein was significantly downgraded, whereas the expression of Bax protein was upregulated in neoplastic tissues. The early apoptotic ratio of 3′‐O‐BOBA‐FUDR‐treated rat group was increased dose‐dependently. These findings strongly support the concept that 3′‐O‐BOBA‐FUDR may be a novel and effective dual‐action antitumor prodrug. Drug Dev Res 72:1–9, 2011. © 2011 Wiley Periodicals, Inc.     

Synthesis of (4‐[18F]fluorophenyl)triphenylphosphonium as a potential imaging agent for mitochondrial dysfunction

Tetraphenylphosphonium (TPP) cation is able to function as a molecular probe for monitoring mitochondrial disease. The F‐18 labeled TPP, (4‐[¹⁸F]fluorophenyl) triphenylphosphonium (¹⁸FTPP), was therefore developed as a PET radioligand for in vivo molecular imaging of mitochondrial dysfunction. ¹⁸FTPP was synthesized via direct nucleophilic substitution of no‐carrier‐added [¹⁸F]fluoride with the precursor 4‐nitrophenyltriphenylphosphonium. After purification by HPLC, the average radiochemical yield was determined to be 10–15% and the specific activity was >500 Ci/mmol at the end of synthesis. The total synthesis time was within 60 min, and the radiochemical purity of the ¹⁸FTPP was above 95%. Copyright © 2005 John Wiley & Sons, Ltd.     

Preparation and biological evaluation of 99mTcN‐4‐(cyclohexylpiperazin‐1‐yl)‐dithioformate as a potential sigma receptor imaging agent

The goal of this study is to develop a novel ^99mTc‐labeled σ receptor imaging agent. Potassium 4‐(cyclohexylpiperazin‐1‐yl)‐dithioformate, 2 , and the corresponding rhenium complex, ReN‐2 , were synthesized and characterized. ReN‐2 possessed moderate affinity toward σ₁ (K_i = 1.94 ± 0.60 µmol/L) and σ₂ (K_i = 2.83 ± 1.39 µmol/L) receptors. The radiolabeled complex ^99m TcN‐2 was prepared in high yield (> 95%) through the [^99mTcN] precursor and characterized by HPLC. ^99m TcN‐2 was found to be a lipophilic and neutral complex with good stability. The biodistribution in tumor‐bearing mice showed that ^99m TcN‐2 had good tumor uptake (2.12 ± 0.01 %ID/g at 2 h p.i.) and moderate brain uptake (0.27 ± 0.05 %ID/g at 2 h p.i.). After blocking with haloperidol, the uptakes by tumor and brain were lower than control. The results indicated that the complex has specific binding to the σ receptors in vivo. Further structural modifications of this complex are needed to obtain ^99mTc‐based σ receptor imaging agents with high affinity and subtype selectivity. Copyright © 2007 John Wiley & Sons, Ltd.     

Synthesis and evaluation of a novel 99mTc nitrido radiopharmaceutical with alendronate dithiocarbamate as a potential bone‐imaging agent

Currently, a popular strategy for designing novel radioprobes as bone‐imaging agents is based on the concept of bifunctional radiopharmaceuticals. Considering the dithiocarbamate ligand can act as a suitable bifunctional linking agent to attach technetium‐99m (^99mTc) to corresponding target molecules, in this study, alendronate dithiocarbamate (ALNDTC) was synthesized and radiolabeled with [^99mTc≡N]²⁺ core by ligand exchange reaction to produce ^99mTcN‐ALNDTC complex, for the potential use as a novel probe for bone imaging. The radiochemical purity of the complex was over 90%. The complex was stable in vitro and could bind to hydroxyapatite. The partition coefficient result indicated it was hydrophilic, and an evaluation of biodistribution in mice indicated that the complex exhibited a higher bone uptake than did ^99mTc‐labeled methylenediphosphonate (^99mTc‐MDP). Further, single photon emission computed tomography imaging study indicated clear accumulation in bone, suggesting that ^99mTcN‐ALNDTC would be a promising candidate for bone imaging.     

Design,Synthesis and Bioevaluation of Novel N‐Substituted‐3,5‐Bis(Arylidene)‐4‐piperidone Derivatives as Cytotoxic and Antitumor Agents with Fluorescent Properties

Ten new N‐substituted‐3,5‐bis(arylidene)‐4‐piperidone derivatives (series 1 and 2 ) were synthesized and subsequently evaluated against human carcinoma cell lines SW1990, MIA PaCa‐2, PG‐BE1, NCI‐H460, and SK‐BR‐3 for cytotoxic activity by the CCK‐8 method, and their fluorescent properties were investigated as well. The compounds were confirmed to display greater cytotoxic activity to the neoplastic cells, and approximately 50% of the IC₅₀ values were lower than 5 μm . In particular, compounds 1a , 1c , 1d, and 1e bearing 3‐bromophenyl groups were revealed as the most active antitumor drug candidates and had the average IC₅₀ values of 1.94, 1.11, 1.16, and 0.817 μm , respectively. Furthermore, their fluorescent properties were interesting and might contribute to the visualization of their distribution in tumor cells. Some possible reasons for the disparity between cytotoxic activity and fluorescent properties in the two series of compounds were explored. This study revealed high potential of these molecules for further development as fluorescent cytotoxic and antitumor agents.     

Synthesis of 2‐[(2‐chloro‐2′‐[18F]fluoroethyl)amino]‐2H‐1,3,2‐oxazaphosphorinane‐2‐oxide (18F‐fluorocyclophosphamide), a potential tracer for breast tumor prognostic imaging with PET

A fluorine‐18 labeled analog of the widely used chemotherapeutic agent cyclophosphamide was synthesized as a tracer for prognostic imaging with positron emission tomography. 2‐[(2‐Chloro‐2′‐[¹⁸F]fluoroethyl)amino]‐2H‐1,3,2‐oxazaphosphorinane‐2‐oxide (¹⁸F‐fluorocyclophosphamide), was prepared by direct halogen exchange reaction from the parent cyclophosphamide. In small‐scale syntheses, radiochemical yields of up to 4.9% and specific activities of 960 Ci/mmol were achieved in a total synthesis time of 60–75 min. The [¹⁸F]‐labeled cyclophosphamide analog with radioactive purity >99% and chemical purity >96% was suitable for in vivo (microPET imaging) and ex vivo studies of a murine model of human breast tumors. Copyright © 2005 John Wiley & Sons, Ltd.     

MKK4 is a novel target for the inhibition of tumor necrosis factor-α-induced vascular endothelial growth factor expression by myricetin

Tumor necrosis factor-α (TNF-α) is a mediator of multiple inflammatory diseases. Vascular endothelial growth factor (VEGF) plays a critical role in TNF-α-mediated diseases. We investigated the inhibitory effects of 3,3′,4′,5,5′,7-hexahydroxyflavone (myricetin), an abundant natural flavonoid, on TNF-α-induced VEGF upregulation and the underlying molecular mechanism. Myricetin is a direct inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) and inhibits neoplastic cell transformation. We found that myricetin inhibited TNF-α-induced VEGF expression in JB6 P+ mouse epidermal cells by targeting MAPK kinase 4 (MKK4), as well as MEK1. The activation of activator protein-1 by TNF-α was inhibited by myricetin in a dose-dependent manner. The phosphorylation of c-Jun N-terminal kinase (JNK) and ERK was inhibited by myricetin, but not the phosphorylation of their upstream kinases MKK4 and MEK1. TNF-α-induced VEGF expression was inhibited by SP600125 and U0126, which are inhibitors of JNK and MEK, respectively. Myricetin inhibited TNF-α-induced MKK4 activity and bound glutathione S-transferase-MKK4 directly by competing with ATP. Computer modeling suggested that myricetin docks onto the ATP-binding site in MKK4, which is located between the N- and C-lobes of the kinase domain. Overall, our results indicate that myricetin has potent chemopreventive effects against TNF-α-related disease, mainly by targeting MKK4 and MEK1.     

Synthesis and evaluation of a new 99mTc(I)‐tricarbonyl complex bearing the 5‐nitroimidazol‐1‐yl moiety as potential hypoxia imaging agent

The objective of this work was to develop a novel ^99mTc complex bearing the 5‐nitroimidazol‐1‐yl moiety with recognised selectivity towards hypoxic tissue, as a potential radiopharmaceutical for imaging tumour hypoxia. The new metronidazole derivative (2‐amine‐3‐[2‐(2‐methyl‐5‐nitro‐1H‐imidazol‐1‐yl)ethylthio]propanoic acid) (L) containing adequate groups to coordinate technetium through the formation of a Tc(I)‐tricarbonyl complex was synthesised with adequate yield (33%) and characterised by spectroscopy. Labelling was performed by substitution of three labile water molecules of the technetium tricarbonyl precursor, fac‐[^99mTc(CO)₃(H₂O)₃]⁺ with the ligand. A radiochemical purity higher than 90% was achieved and remained unchanged for more than 4 h. The complex has a high stability in plasma, a moderate plasma protein binding and a moderate hydrophilicity. In vitro cell uptake studies showed a ratio between the activity taken up by cells in hypoxia/normoxia of 1.6 ± 0.4 (p < 0.5). Biodistribution in normal mice showed rapid depuration and low uptake in all organs and tissues except liver. Biodistribution in mice bearing induced tumours showed a low tumour uptake, but tumour/muscle ratio was favourable thanks to depuration. Comparison with biological results of other metronidazole derivatives clearly shows that modifications of the chelator are very important and contribute to improve the biological behaviour.     

Synthesis of 18F‐labeled cyclooxygenase‐2 (COX‐2) inhibitor as a potential PET imaging agent

A new PET tracer for COX‐2 imaging, the 6‐ethoxy‐3‐(4‐methanesulfonylphenyl)‐4‐(4‐[¹⁸F]fluorophenyl)pyran‐2‐one ([¹⁸F]EFMP), was synthesized. For F‐18 radiolabeling, a trimethylammonium precursor and a brominated precursor were synthesized from 1,1,2,3‐tetrachlorocycloprop‐2‐ene in 6 steps. The radiolabeling was achieved through nucleophilic substitution using no‐carrier‐added (n.c.a.) fluorine‐18. Solid‐phase extraction and semi‐preparative‐HPLC purification produced [¹⁸F]EFMP in 14.6±3.3% (n =4) decay corrected radiochemical yield with a specific activity of 487±85.1 (n =4) Ci/mmol and greater than 98% radiochemical purity. Copyright © 2006 John Wiley & Sons, Ltd.     

Synthesis and 124I‐labeling of m‐iodophenylpyrrolomorphinan as a potential PET imaging agent for delta opioid (DOP) receptors

Condensation of phenylazo‐β‐ketoamide 4 with oxymorphone 5 afforded an m‐iodophenylpyrrolomorphinan (m‐IPPM) 6 mediated by elemental zinc in acetic acid/sodium acetate buffer. m‐IPPM 6 is a novel opioid receptor agonist (K_i = 4.53 nM for DOP) with high selectivity for DOP receptors. m‐IPPM 6 was converted into the positron emitter m‐[¹²⁴I]PPM 8 via the stannylated intermediate 7 . The final yield was 24.5 ± 1.9 % (n = 6) with a specific activity of 2.5 ± 1.2 Ci/µmol. Copyright © 2007 John Wiley & Sons, Ltd.     

Synthesis of an analogue of α-MSH-(1–10)-decapeptide as a substrate for enzymatic Nα-acetylation

The synthesis of a stable substrate for enzymatic N^α-acetylation is described. To this end an analogue of α-MSH-(1–10)-decapeptide with norleucine instead of methionine at position 4 is prepared. t-Butylation of an intermediate Z-Tyr-OMe leads only to about 75% conversion in a few hours' reaction time, and cannot be carried to completion. The [Nle⁴]-decapeptide is as good a substrate for enzymatic N^α-acetylation as the original decapeptide containing methionine.     

Macrocyclic peptides II. Synthesis and structure of a novel dipeptide, (2S,3′S)-2-(2′-oxo-3′-methylpiperazin-1′-yl)-propanoic acid,and its use as the unit of cyclic peptides

A new cyclic compound [(2S,3′S)-2-(2′-oxo-3′-methylpiperazin-1′-yl)-propanoic acid] obtained using (2S,7S)-2,7-dimethyl-3,6-diazaoctanedioic acid as a starting material was derived from N-t-butyloxycarbonyl-(2S,3′S)-2-(2′-oxo-3′-methylpiperazin-1′-yl)-propanoic acid in order to determine the molecular structure by X-ray crystal analysis. Two new macrocyclic peptides were prepared using the above cyclic compound and sarcosine as peptide units.