Cornelis Marius Timmers

The First Orally Active Low Molecular Weight Agonists for the LH Receptor: Thienopyr(im)idines with Therapeutic Potential for Ovulation Induction

The gonadotropins, a class of glycoproteins with an average molecular weight of 30 kD, play a pivotal role in human reproduction. Follicle stimulating hormone (FSH), for example, causes ovarian follicle maturation in women and is involved in spermatogenesis in men. Luteinizing hormone (LH) is responsible for ovulation induction in women and controls testosterone production in men. Finally, human choriogonadotropin (hCG) maintains the early stages of a pregnancy. All gonadotropins consist of a common subunit and a hormone-specific Support from the Forschungsinstitut f ̧r Molekulare Pharmakologie is gratefully acknowledged. M.S. was supported by the DFG Graduiertenkolleg GRK 80 TMModellstudien∫. The authors thank Wolfgang Bermel for helpful discussions regarding the setup of the TROSY experiments, Hartmut Oschkinat for his continuous encouragement, and Karen Zierler for carefully reading the manuscript.

Induction of ovulation by a potent, orally active, low molecular weight agonist (Org 43553) of the luteinizing hormone receptor

BACKGROUND In assisted reproductive technology, human chorionic gonadotrophin (hCG) is administered subcutaneously for the induction of oocyte maturation and ovulation. Our efforts to develop orally bioavailable luteinizing hormone (LH) receptor agonists have led to the discovery of Org 43553, a low molecular weight (LMW) LH receptor (LH-R) agonist. METHODS Org 43553 was tested in vitro and in vivo in pre-clinical pharmacological models to demonstrate efficacy and oral availability. RESULTS Org 43553 is a potent stimulator of the human LH-R in vitro (EC(50) 3.7 nM). In primary mouse Leydig cells, Org 43553 stimulated testosterone production. Pharmacokinetic analyses showed high oral bioavailability in rats (79%) and dogs (44%) with a shorter half-life compared with hCG (3.4 versus 5.6 h in the rat). Ovulation induction by Org 43553 was demonstrated in immature mice as well as in cyclic rats after single-dose oral administration (50 mg/kg). The ovulated oocytes were of good quality as demonstrated by successful fertilization and implantation of normal embryos. In male rats, testosterone production was substantially induced after oral administration. CONCLUSIONS Org 43553 is the first LMW LH-R mimetic with demonstrated in vivo efficacy upon oral administration and could therefore replace subcutaneously administered hCG. The elimination half-life of Org 43553 is substantially shorter than hCG, which could potentially represent a clinical benefit in reducing the risk of ovarian hyperstimulation syndrome (OHSS).

[3H]Org 43553, the First Low Molecular Weight Agonistic and Allosteric Radioligand for the Human Luteinizing Hormone Receptor

The luteinizing hormone (LH) receptor plays a pivotal role in reproduction. The high-molecular-weight (HMW) human chorionic gonadotropin (hCG) and LH are the endogenous ligands of this receptor and bind to its large N terminus. The present study characterizes the binding of a new low-molecular-weight (LMW) radioligand, [3H]5-amino-2-methylsulfanyl-4-[3-(2-morpholin-4-yl-acetylamino)-phenyl]-thieno[2,3-d]pyrimidine-6-carboxylic acid tert-butylamide (Org 43553), at the LH receptor. Equilibrium saturation and displacement assays were developed and optimized. Specific binding of [3H]Org 43553 to CHO-K1 cell membranes expressing the human LH receptor and a cAMP response element-luciferase reporter gene was saturable with a KD value of 2.4 ± 0.4 nM and a Bmax value of 1.6 ± 0.2 pmol/mg protein. Affinities of five LMW analogs of Org 43553 were determined. All displaced the radioligand competitively, with Ki values ranging from 3.3 to 100 nM. Finally, the potency of these compounds in a cAMP-induced luciferase assay was also determined. There was a high correlation between affinity and potency (r = 0.99; P < 0.0001) of these compounds. In the search for LMW ligands, which bind allosterically to the seven-transmembrane domain of the LH receptor, a HMW radioligand (e.g., 125I-hCG) is not suitable as it is not displaced by a LMW compound. Therefore, [3H]Org 43553, a new radioligand with good binding properties, allows screening for new LMW ligands that mimic the action of the endogenous hormone at the LH receptor.

Rhodium(II) catalyzed asymmetric cyclopropanation of glycals with ethyl diazoacetate

Abstract Carbenoid species generated from ethyl diazoacetate and catalytic Rh 2 (OAc) 4 react smoothly and with a high degree of stereoselectivity with glycals resulting in the predominant formation of doubly branched adducts containing an α- exo -orientated ethyl cyclopanecarboxylate moiety.

Stereoselective Synthesis of α-C-(alkynyl)-glycosides via Ring-opening of α-1,2-Anhydrosugars

Ring-opening of an α-1,2-epoxide function in sugars with lithium alkynyl derivatives in the presence of zinc chloride proceeds with retention of configuration to afford α-C-(alkynyl)-glycosides in reasonable yields.

Identification of an altered peptide ligand based on the endogenously presented, rheumatoid arthritis-associated, human cartilage glycoprotein-39(263–275) epitope: an MHC anchor variant peptide for immune modulation

We sought to identify an altered peptide ligand (APL) based on the endogenously expressed synovial auto-epitope of human cartilage glycoprotein-39 (HC gp-39) for modulation of cognate, HLA-DR4-restricted T cells. For this purpose we employed a panel of well-characterized T cell hybridomas generated from HC gp-39-immunized HLA-DR4 transgenic mice. The hybridomas all respond to the HC gp-39(263–275) epitope when bound to HLA-DR4(B1*0401) but differ in their fine specificities. First, the major histocompatibility complex (MHC) and T-cell receptor (TCR) contact residues were identified by analysis of single site substituted analogue peptides for HLA-DR4 binding and cognate T cell recognition using both T hybridomas and polyclonal T cells from peptide-immunized HLA-DR4 transgenic mice. Analysis of single site substituted APL by cognate T cells led to identification of Phe265 as the dominant MHC anchor. The amino acids Ala268, Ser269, Glu271 and Thr272 constituted the major TCR contact residues, as substitution at these positions did not affect HLA-DR4(B1*0401) binding but abrogated T cell responses. A structural model for visualisation of TCR recognition was derived. Second, a set of non-classical APLs, modified at the MHC key anchor position but with unaltered TCR contacts, was developed. When these APLs were analysed, a partial TCR agonist was identified and found to modulate the HC gp-39(263–275)-specific, pro-inflammatory response in HLA-DR4 transgenic mice. We identified a non-classical APL by modification of the p1 MHC anchor in a synovial auto-epitope. This APL may qualify for rheumatoid arthritis immunotherapy.

Synthesis and Pharmacological Evaluation of Dimeric Follicle-Stimulating Hormone Receptor Antagonists

A series of homo‐ and heterodimeric compounds encompassing the follicle‐stimulating hormone receptor (FSHR) antagonist (R)‐1 and its inactive conformer (S)‐1 connected through ethylene glycol spacers of various lengths is described. Evaluation of these compounds reveals that dimeric compounds, with a spacer of sufficient length, bearing two active copies of the antagonist are more potent relative to dimeric compounds in which one of the active pharmacophores is replaced by an inactive conformer. Interestingly, the opposite trend is observed if a short spacer is used, indicating that these compounds may be valuable tools to study FSHR dimerization in greater detail.

Synthesis and evaluation of homodimeric GnRHR antagonists having a rigid bis-propargylated benzene core

The fact that GPCRs might function in a dimeric fashion is currently well accepted. For GnRHR, a GPCR that regulates gonadotropin release, there is evidence that the receptor also functions as a dimer. We here describe the design and synthesis of a set of dimeric GnRHR antagonists in order to understand the interaction of dimeric ligands to the receptor and to address the question whether GnRHR dimerization is a prerequisite for signalling. Biological evaluation of the compounds shows no discrimination between monomeric and dimeric-ligands in respect to binding affinities, however, the dimeric ligands appear to have different functional properties.

Discovery of selective luteinizing hormone receptor agonists using the bivalent ligand method.

Two series of dimeric ligands for a G‐protein‐coupled receptor were prepared that differ by the interconnecting spacer system. Biological evaluation revealed that both dimeric series exhibit unique biological properties relative to their monomeric counterparts.

A Study Towards Total Synthesis of Antibiotic Agrocin 84

Abstract A versatile synthetic route to compound 2 - an analog of the naturally occurring bacteriocin Agrocin 84 is described.