Synthesis and applications of cyclopeptides and depsipeptides

Summary A solid phase protocol has been devised for the synthesis of linear precursors to cyclic depsipeptide analogues of dolastatin D.t-Butyldimethylsilyl groups were used for hydroxy group protection, with deprotection being carried out byt-butyl ammonium fluoride. HATU and PyBrop were successful in coupling highly hindered residues and in depside bond formation. Cyclic peptide analogues, cyclo[Arg-Gly-Asp-d-Phe-Lys(or Tyr)] have been synthesised and modified for use as carrier molecules for the transport of radio isotopes (¹¹¹In and¹²⁵I) into blood platelets as prototypes for medical imaging.     

Sulfonamide phenylalanine (SPA) series of analogues as an antibacterial,antifungal, anticancer agents along with p53 tumor suppressor-DNA complex inhibitor – part 1

Abstract

A series of N-[1-benzyl-2-oxo-2-substituted(ethyl)] benzene/p-toluene sulfonamide (K1–K12) are synthesized. Structure of the synthesized analogues has been confirmed by FT-IR, ¹H & ¹³C NMR and ESI-MS spectroscopic techniques. All the synthesized analogues (K1–K12) have also been examined for their in-vitro antibacterial and antifungal activities. Compounds showed good antibacterial and antifungal activity against standard drug. Anticancer study has been carried out on three cancer cell lines PC-3, MCF-7 and A549 on two different concentrations (mg/mL and μg/mL). The K4 sulfonamide analogue showed better anticancer activity amongst all analogues against PC-3 and A549 cell lines. K4 inhibit G0/G1 phase in cell-cycle analysis experiment. All synthesized molecules (K1–K12) dock at junction p53-DNA and make hydrogen bonded with residues of p53 protein as per docking study. ADMET predictions of synthesized phenylalanine sulfonamide analogues (K1–K12) has been done using ‘Lipinski rule’ and it has been observed that all synthesized analogues did not violate the rule. Electronic, chemical properties and mulliken atomic charges of analogues were calculated using density functional theory (DFT).

Communicated by Ramaswamy H. Sarma     

Action of cyclic nucleotide analgoues in Chinese hamster ovary cells

Cyclic nucleotide analogues have been tested for their ability to cause the morphological conversion of Chinese hamster ovary cells in culture, as well as for effects on cyclic AMP-related enzymes. The ability of the analogues to inhibit the cyclic AMP phosphodiesterase activity and to activate the cyclic AMP-dependent protein kinase activity in cell extracts has been measured. Cell cultures were incubated with the analogues and the effects on morphology, intracellular level of cyclic AMP, and in vivo protein kinase activation were determined. All analogues which induced the morphological conversion also caused in vivo activation of the cyclic AMP-dependent protein kinase. Only N⁶,O^2′-dibutryl and N⁶-monobutyryl cyclic AMp caused caused on increase in intracellular cyclic AMP, presumably through inhibition of the intracellular cyclic AMP phosphodiesterase activity. The increase in cyclic AMP appears to cause the protein kinase activation. However, analogues such as 8-bromo and 8-benzylthio cyclic AMP do not cause any change in intracellular cyclic AMP level and appear to activate the intracellular cyclic AMP-dependent protein kinase directly.     

Structural elucidation of Leuprolide and its analogues in solution: insight into their bioactive conformation

Leuprolide [dLeu⁶, NHEt¹⁰]GnRH, a potent gonadotropin-releasing hormone (GnRH) agonist, is used in a wide variety of hormone-related diseases like cancer and endometriosis. In this report, the conformational behaviour of Leuprolide and its linear synthetic analogues, namely [Tyr⁵(OMe), dLeu⁶, Aze⁹, NHEt¹⁰]GnRH (1) and [Tyr⁵(OMe), dLeu⁶, NHEt¹⁰]GnRH (2) have been studied in DMSO and H₂O solutions by means of 2D nuclear magnetic resonance (NMR) experiments and detailed molecular dynamics (MD) simulations. The aim was to identify the conformational requirements of GnRH analogues for agonistic activity. This approach is of value as no crystallographic data are available for the GnRH receptor (G protein-coupled receptor, GPCR). The NOE data indicate the existence of a β-turn type I in the 2–5 segments of Leuprolide and its linear analogues in the case of using DMSO-d₆ as solvent, whereas a β-turn type II in the 3–6 segments is indicated using D₂O as solvent. The final structures fulfil the conformational requirements that are known, in the literature, to play a significant role in receptor recognition and activation. Finally, the linear analogues (1) and (2) are biologically active when tested against the human breast cancer cell line, MCF-7.     

Synthesis and Biological Evaluation of New GnRH Analogues on Pituitary and Breast Cancer Cells

GnRH analogues have been extensively used in oncology to induce reversible chemical castration due to their hypophysiotropic action. In addition to that, it has recently been shown that many malignant cells, such as breast cancer cells, locally produce GnRH and express the GnRH receptor/s. In order to investigate the structure-activity relationships in both pituitary and extrapituitary biological systems, we synthesized eight new GnRH analogues with modifications in the N-terminal part and/or in position 6 and studied their pituitary binding affinity (in αT3-1 cell membranes) and effect on breast cancer (MCF-7) cell proliferation. 2-Amino-4-pyrrolidinothieno[2,3-d]pyrimidine-6-carboxylic acid (ATPC) was incorporated instead of pGlu¹-His²- and/or Gly⁶ was substituted by α-aminoisobutyric acid, D-Leu and D-Lys (alone or covalently linked to Gly, Ala, Sar, ATPC). Most GnRH analogues lacked the carboxy-terminal Gly¹⁰-amide of GnRH and an ethylamide residue was added to Pro⁹, a modification common in many potent GnRH agonists, such as leuprolide ([D-Leu⁶, des-Gly¹⁰]-GnRH-NHEt. Results show differential impact of these modifications on the binding affinity to the GnRH receptor in mouse pituitary cells and on the inhibition of human breast cancer cell proliferation. ATPC in the N-terminus resulted in analogues with low binding affinity but high antiproliferative effect. Substitutions in position 6 always resulted in high binding affinities. In particular, [D-Lys⁶(Gly), desGly¹⁰]-GnRH-NHEt and [D-Lys⁶(Sar), desGly¹⁰]-GnRH-NHEt have higher pituitary binding affinity than leuprolide, but only the latter had significant antiproliferative effect on both MCF-7 and MDA-MB-231 cells. These results contribute to the on-going research for more potent GnRH analogues. Abbreviations of common amino acids are in accordance with the recommendations of IUPAC-IUB Joint Commission on Biochemical Nomenclature: Arch. Biochem. Biophys. 206, pp.v-xxii (1988), J. Biol. Chem. 264, 668–673 (1989) or J. Peptide Sci. 9, 1–8 (2003).     

Synthesis,thymidine phosphorylase,angiogenic inhibition and molecular docking study of isoquinoline derivatives

Isoquinoline analogues (KA-1 to 16) have been synthesized and evaluated for their E. coli thymidine phosphorylase inhibitory activity. Except compound 11, all other analogs showed outstanding thymidine inhibitory potential ranging in between 4.40 ± 0.20 to 69.30 ± 1.80 µM when compared with standard drug 7-Deazaxanthine (IC₅₀ = 38.68 ± 4.42 µM). Structure Activity Relationships has been established for all compounds, mainly based on substitution pattern on phenyl ring. All analogs were characterized by various spectroscopic techniques such as ¹H NMR, ¹³C NMR and EI-MS. The binding interactions of isoquinoline analogues with the active site of TP enzyme, the molecular docking studies were performed. Furthermore, the angiogenic inhibitory potentials of isoquinoline analogues (KA-1-9, 14, 12 and 16) were determined in the presence of standard drug Dexamethasone based on percentage inhibitions at various concentrations. Herein this work analogue KA-12, 14 and 16 emerged with most potent angiogenic inhibitory potentials among the synthesized analogues.     

Linear and Cyclic α-Melanotropin [4–10]-Fragment Analogues That Exhibit Superpotency and Residual Activity

Two analogues of α-MSH (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂), Ac-[Nle⁴, Asp⁵, D-Phe⁷, Lys¹⁰]α-MSH_4–10NH₂ and Ac-[Nle⁴, Asp⁵, D-Phe⁷, Lys¹⁰] α-MSH_4–10-NH₂, were synthesized, and the melanotropic activities of the peptides were compared in several bioassays. Potencies were determined in the in vitro frog and lizard skin bioassays and in the S91 melanoma cell tyrosinase assay. Both analogues were equipotent or more potent than α-MSH in all bioassays, and the activities of the analogues were prolonged compared to α-MSH. The two analogues were very resistant to inactivation by purified proteolytic enzymes (α-chymotrypsin, trypsin, and pepsin). The two peptides could be topically applied and transdermally delivered across the skin of mice in vivo, resulting in a shift from pheomelanogenesis to eumelanogenesis within follicular melanocytes. The cyclic analogue exhibited greater potency, prolonged activity, and stability against enzyme inactivation than did the linear peptide. The significance of the findings for the further design of melanotropin analogues is discussed, as in the possible relevance of these melanotropin analogues for use in biomedical studies.     

Synthesis and GABAA receptor activity of 2,19-sulfamoyl analogues of allopregnanolone

The synthesis of new analogues of allopregnanolone with a bridged sulfamidate ring over the β-face of ring A has been achieved from easily available precursors, using an intramolecular aziridination strategy. The methodology also allows the synthesis of 3α-substituted analogues such as the 3α-fluoro derivative. GABA_A receptor activity of the synthetic analogues was evaluated by assaying their effect on the binding of [³H]flunitrazepam and [³H]muscimol. The 3α-hydroxy-2,19-sulfamoyl analogue and its N-benzyl derivative were more active than allopregnanolone for stimulating binding of [³H]flunitrazepam. For the binding of [³H]muscimol, both synthetic analogues and allopregnanolone stimulated binding to a similar extent, with the N-benzyl derivative exhibiting a higher EC₅₀. The 3α-fluoro derivative was inactive in both assays.     

Synthesis and in vitro evaluation of [18F]fluoroethyl triazole labelled [Tyr3]octreotate analogues using click chemistry

A novel class of alkyne linked [Tyr³]octreotate analogues have been labelled by a copper catalysed azide-alkyne cycloaddition reaction (CuAAC) to form a 1,4-substituted triazole using the reagent [¹⁸F]2-fluoroethyl azide. An unexpected variability in reactivity during the CuAAC reaction was observed for each alkyne analogue which has been investigated. Two lead alkyne linked [Tyr³]octreotate analogues, G-TOCA (3a) and βAG-TOCA (5a) have been identified to be highly reactive in the click reaction showing complete conversion to the [¹⁸F]2-fluoroethyl triazole linked [Tyr³]octreotate analogues FET-G-TOCA (3b) and FET-βAG-TOCA (5b) under mild conditions and with short synthesis times (5 min at 20 °C). As well as ease of synthesis, in vitro binding to the pancreatic tumour AR42J cells showed that both FET-G-TOCA and FET-βAG-TOCA have high affinity for the somatostatin receptor with IC₅₀ of 4.0 ± 1.4, and 1.6 ± 0.2 nM, respectively.     

I. The development of amine substituted analogues of MPTP as unique tools for the study of MPTP toxicity and Parkinson's disease

We are currently developing amino-substituted MPTP analogues as useful probes for understanding the mechanism of MPTP toxicity and Parkinson's disease. One analogue, 4′-amino MPTP, induces a loss of striatal dopamine and is thus a suitable substitute for MPTP. This probe will be used as a histologically fixable MPTP which can be used to answer detailed anatomical questions concerning the sites of MPTP, MPP⁺ uptake and storage. In addition, antibodies have been raised against MPTP and MPP⁺ in rabbits using diazo-linked bovine serum albumin conjugates. The antibodies have been characterized with regard to their recognition of relevant structural analogues using an enzymelinked immunoassay (ELISA) procedure. Antibodies to MPTP detected MPTP in mouse brain extracts derived from as little as 5 μg of tissue. The antibodies will be used for immunohistochemical localization of 4′-NH₂-MPTP and 4′-NH₂-MPP⁺ in brain, as well as probes for the screening of parkinsonian brain tissue for any MPTP- or MPP⁺-like materials which might exist.     

Real-time NMR Study of Guanine Nucleotide Exchange and Activation of RhoA by PDZ-RhoGEF

Small guanosine triphosphatases (GTPases) become activated when GDP is replaced by GTP at the highly conserved nucleotide binding site. This process is intrinsically very slow in most GTPases but is significantly accelerated by guanine nucleotide exchange factors (GEFs). Nucleotide exchange in small GTPases has been widely studied using spectroscopy with fluorescently tagged nucleotides. However, this method suffers from effects of the bulky fluorescent moiety covalently attached to the nucleotide. Here, we have used a newly developed real-time NMR-based assay to monitor small GTPase RhoA nucleotide exchange by probing the RhoA conformation. We compared RhoA nucleotide exchange from GDP to GTP and GTP analogues in the absence and presence of the catalytic DH-PH domain of PDZ-RhoGEF (DH-PH^PRG). Using the non-hydrolyzable analogue guanosine-5′-O-(3-thiotriphosphate), which we found to be a reliable mimic of GTP, we obtained an intrinsic nucleotide exchange rate of 5.5 × 10⁻⁴ min⁻¹. This reaction is markedly accelerated to 1179 × 10⁻⁴ min⁻¹ in the presence of DH-PH^PRG at a ratio of 1:8,000 relative to RhoA. Mutagenesis studies confirmed the importance of Arg-868 near a conserved region (CR3) of the Dbl homology (DH) domain and revealed that Glu-741 in CR1 is critical for full activity of DH-PH^PRG, together suggesting that the catalytic mechanism of PDZ-RhoGEF is similar to Tiam1. Mutation of the single RhoA (E97A) residue that contacts the pleckstrin homology (PH) domain rendered the mutant 10-fold less sensitive to the activity of DH-PH^PRG. Interestingly, this mutation does not affect RhoA activation by leukemia-associated RhoGEF (LARG), indicating that the PH domains of these two homologous GEFs may play different roles.     

Ivermectin-derived leishmanicidal compounds

In the present study a family of macrocyclic and acyclic analogues as well as seco-analogues of avermectins were prepared from commercial Ivermectin (IVM) and their antileishmanial activity assayed against axenic promastigote and intracellular amastigote forms of Leishmania amazonensis. Contrarily to the filaricidal activity, the leishmanicidal potentiality of avermectin analogues does not appear to depend on the integrity of the non-conjugated Δ^3,4-hexahydrobenzofuran moiety. Conjugated Δ^2,3-IVM or its corresponding conjugated secoester show higher anti-leishmania activity than the parent compound. Surprisingly, the diglycosylated northern sub-unit exhibits the same anti-amastigote potentiality as the southern hexahydrobenzofuran. As expected for compounds derived from the widely used Ivermectin antibiotic, little toxicity has been noticed for most of the novel analogues prepared.     

Synthesis and bioactivity of chemotactic tetrapeptides: fMLF-OMe analogues incorporating spacer aminoacids at the lateral positions

A small library of N-For and N-Boc tetrapeptidic analogues of the chemotactic tripeptide For-Met-Leu-Phe-OMe (fMLF-OMe), obtained by incorporating three different spacer aminoacids (Gly, βAla and Pro) between the native residues of Met and Leu (N-For- and N-Boc-Met-Xaa-Leu-Phe-OMe; Xaa² series) and Leu and Phe (N-For- and N-Boc-Met-Leu-Xaa-Phe-OMe; Xaa³ series), have been synthesized and examined for their biological activity as agonists and antagonists. Chemotaxis, lysozyme release and superoxide anion production have been measured. All the N-For analogues maintain good to moderate chemotactic activity with the βAla³ 15 model reaching the maximum value. All the N-Boc tetrapeptides are efficient chemotactic antagonists. Conversely, with the exception of the moderate antagonistic activity exhibited by the N-Boc Xaa² models against lysozyme release, all the other N-Boc analogues do not show significant activity against both superoxide anion and lysozyme release.     

Synthesis and Biological Activity of Modified Thiopyrimidine Nucleosides

Abstract

N³ -β-D-glucopyranosyl, galactopyranosyl and xylopyranosyl 6-methyl-2-methylthiouracil and their 5-bromo derivatives have been synthesized by coupling an a-acetobromosugar with the corresponding thiouracil. The new modified thiouridine analogues were evaluated for their inhibitory activity against Human Immunodeficiency Virus (HIV) replication in MT-4 cells as well as for their cytotoxicity.     

Synthesis and Properties of Mixed Anhydrides of AMP,ADP and ATP with Mesitoic Acid

Abstract

New affinity reagents for ATP-dependent enzymes are described. Optimal conditions are evolved for the synthesis of mixed anhydrides of AMP, ADP, ATP with mesitoic acid (MsCOp_nA, n = 1?3) and for their 1, N⁶-etheno, 2′, 3′-dialdehyde and photoactive analogues. UV, CD and fluorescence spectra of the compounds have been analyzed. Hydrolysis of MsCOp_nA (n = 1?3) and their etheno analogues over a wide pH range has been carried out.     

Synthesis of new N-(arylcyclopropyl)acetamides and N-(arylvinyl)acetamides as conformationally-restricted ligands for melatonin receptors

N-(Arylcyclopropyl)acetamides and N-(arylvinyl)acetamides or methyl ureas have been prepared as constrained analogues of melatonin. The affinity of these new compounds for chicken brain melatonin receptors and recombinant human MT₁ and MT₂ receptors was evaluated using 2-[¹²⁵I]-iodomelatonin as radioligand. Strict ethylenic or cyclopropyl analogues of the commercialized agonist agomelatine (Valdoxan®) were equipotent to agomelatine in binding bioassays. However, the ethylenic analogue was more effective than the cyclopropyl one in the melanophore aggregation bioassay, but was still less potent than the disubstituted 2,7-dimethoxy-naphtalenic compounds.     

Synthesis of new vasotocin analogues: effects on renal water and ion excretion in rats

Vasopressin and nonmammalian hormone vasotocin are known to increase the water permeability of mammalian collecting ducts, frog skin and the urinary bladder. Neurohypophysial nonapeptides have also been shown to interfere with the regulation of renal ion transport. The subject of this study was a search for vasopressin and vasotocin analogues with selective effects on renal water, sodium and potassium excretion. During this study, we synthesised the following peptides: 13 vasotocin analogues modified at positions 4 (Thr or Arg), 7 (Gly or Leu) and 8 (d ‐Arg, Lys or Glu); 4 vasopressin analogues modified at positions 4 and 8; and 9 peptides shortened or extended at the C‐terminal or with substitutions for Gly‐NH₂. Most of these peptides had mercaptopropionic acid (Mpa) instead of Cys in position 1. The effects of these nonapeptides on renal water, sodium and potassium transport were evaluated in in vivo experiments using Wistar rats. Some nonapeptides possessed antidiuretic, natriuretic and kaliuretic activities ([Mpa¹]‐arginine vasotocin, [Mpa¹, homoArg⁸]‐vasotocin, [Mpa¹, Thr⁴]‐arginine vasotocin and [Mpa¹, Arg⁴]‐arginine vasopressin). Substitutions at positions 4 and 8 increased the selectivity of peptide actions. The antidiuretic [d ‐Arg⁸]‐vasotocin analogues had no effects on sodium excretion. [Mpa¹, Arg⁴]‐arginine vasotocin was antidiuretic and kaliuretic but not natriuretic. [Mpa¹, Glu⁸]‐oxytocin had weak natriuretic activity without any effects on water and potassium transport. In accordance with the data obtained, synthesised vasotocin analogues could be good candidates for pharmaceuticals selectively regulating renal sodium and potassium transport, which is of clinical importance. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Site-directed affinity-labeling of delta opioid receptors by

The S-3-nitro-2-pyridinesulfenyl (SNpys) group in an affinity ligand can bind to a free thiol group of a cysteine residue in a target receptor molecule, forming a disulfide bond via the thiol-disulfide exchange reaction. SNpys-containing Leu-enkephalin analogues of [-Ala², Leu⁵]-enkephalyl-Cys(Npys)⁶ and [-Ala²,Leu(CH₂SNpys)⁵]enkephalin, and dynorphin A analogues of [-Ala²,Cys(Npys)¹²]dynorphin A-(1-13) amide and [-Ala²,Cys(Npys)⁸]dynorphin A-(1-9) amide have been found to affinity-label all of the δ, μ (rat brain), and κ (guinea pig brain) opioid receptor subtypes. In this study, using these chemically synthesized SNpys-containing analogues, we attempted to identify the analogues that affinity-label the cysteine residue at position 60 of the δ opioid receptor. We first established the assay procedure, principally based on the receptor binding assay to use COS-7 cells expressing the δ opioid receptor. Then, using a mutant δ receptor with the Cys60→Ala substitution, we assayed the SNpys-containing analogues for their specific affinity-labeling. [-Ala²,Cys(Npys)¹²]dynorphin A-(1-13) amide was found to have drastically reduced labeling activity for this mutant receptor as compared to its activity for the wild-type δ receptor. Other analogues exhibited almost the same activity for both the wild-type and mutant δ receptors. These results indicate that the δ-Cys60 residue has a free thiol group, which is labeled by [-Ala²,Cys(Npys)¹²]dynorphin A-(1-13) amide.     

Spectral analysis of a series of partially protected and deprotected tetrapeptides, analogues of AS-I phytotoxin

Summary A series of six tetrapeptides, analogues of AS-I phytotoxin, pathogenic to sunflower, have been synthesized either in solution and/or by solid phase methods and have been tested for phytotoxic activity in various plants and cytotoxic activity in three cancer cell lines. These peptides were identified as model compounds by fast atom bombardment (FAB), plasma desorption (PD), electrospray ionization (ESI) mass spectrometry and by¹H,¹H-¹H,¹³C and¹H-¹³C NMR. The data presented show that in protected tetrapeptides the molecular ion was easily identified whereas some difficulties appeared with the fully deprotected peptides. NMR spectra are given.     

Chemical Scale Studies of the Phe-Pro Conserved Motif in the Cys Loop of Cys Loop Receptors

The functions of two conserved residues, Phe¹³⁵ and Pro¹³⁶, located at the apex of the Cys loop of the nicotinic acetylcholine receptor are investigated. Both residues were substituted with natural and unnatural amino acids, focusing on the role of aromaticity at Phe¹³⁵, backbone conformation at Pro¹³⁶, side chain polarity and volume, and the specific interaction between the aromatic side chain and the proline. NMR spectroscopy studies of model peptides containing proline and unnatural proline analogues following a Phe show a consistent increase in the population of the cis conformer relative to peptides lacking the Phe. In the receptor, a strong interaction between the Phe and Pro residues is evident, as is a strong preference for aromaticity and hydrophobicity at the Phe site. A similar influence of hydrophobicity is observed at the proline site. In addition, across a simple homologous series of proline analogues, the results reveal a correlation between receptor function and cis bias at the proline backbone. This could suggest a significant role for the cis proline conformer at this site in receptor function.