Anja Hoffmann-Röder

Predicting and Tuning Physicochemical Properties in Lead Optimization: Amine Basicities

This review describes simple and useful concepts for predicting and tuning the pKa values of basic amine centers, a crucial step in the optimization of physical and ADME properties of many lead structures in drug‐discovery research. The article starts with a case study of tricyclic thrombin inhibitors featuring a tertiary amine center with pKa values that can be tuned over a wide range, from the usual value of around 10 to below 2 by (remote) neighboring functionalities commonly encountered in medicinal chemistry. Next, the changes in pKa of acyclic and cyclic amines upon substitution by fluorine, oxygen, nitrogen, and sulfur functionalities, as well as carbonyl and carboxyl derivatives are systematically analyzed, leading to the derivation of simple rules for pKa prediction. Electronic and stereoelectronic effects in cyclic amines are discussed, and the emerging computational methods for pKa predictions are briefly surveyed. The rules for tuning amine basicities should not only be of interest in drug‐discovery research, but also to the development of new crop‐protection agents, new amine ligands for organometallic complexes, and in particular, to the growing field of amine‐based organocatalysis.

A fluorine scan at the catalytic center of thrombin: C--F, C--OH, and C--OMe bioisosterism and fluorine effects on pKa and log D values.

A series of 16 tricyclic thrombin inhibitors was prepared by using the 1,3‐dipolar cycloaddition of azomethine ylides derived from 3‐ or 4‐hydroxyproline and 4‐bromobenzaldehyde, with N‐(4‐fluorobenzyl)maleimide as the key step. The terminal pyrrolidine ring of the inhibitors was systematically substituted to explore the potential bioisosteric behavior of Cuf8ffF, Cuf8ffOH, and Cuf8ffOMe residues pointing into the environment of the catalytic center of a serine protease. X‐ray crystal structure analyses revealed a distinct puckering preference of this ring. Substitution by F, HO, and MeO has a strong effect on the basicity of the adjacent pyrrolidine nitrogen center which originates from two σ‐inductive pathways between this center and the electronegative O and F atoms. gem‐Difluorination decreases the pKa value of this tertiary amine center to <2, making the conjugated ammonium ion a moderately strong acid. Unexpectedly, F substitution next to the nitrogen center reduced the lipophilicity of the ligands, as revealed by measurements of the logarithmic partition coefficient logu2009D. The biological assays showed that all compounds are thrombin inhibitors with activities between Ki=0.08 and 2.17u2005μM. Bioisosteric behavior of F, HO, and MeO substituents was observed. Their electronegative F and O atoms undergo energetically similar polar interactions with positively polarized centers, such as the Nu2005atom of Hisu200957 which is hydrogen bonded to the catalytic Seru2009195. However, for energetically similar polar interactions of Cuf8ffF, Cuf8ffOH, and Cuf8ffOMe to occur, sufficient space is necessary for the accommodation of the Me group of the Cuf8ffOMe residue, and a H‐bond acceptor must be present to prevent unfavorable desolvation of the Cuf8ffOH residue.

Synthesis of a MUC1-glycopeptide-BSA conjugate vaccine bearing the 3'-deoxy-3'-fluoro-Thomsen-Friedenreich antigen.

A novel MUC1-glycopeptide-BSA conjugate vaccine with a specifically fluorinated Thomsen-Friedenreich antigen side chain at Thr6 was prepared. Preliminary immunological experiments reveal specific binding of the tumor-associated glycopeptide antigen analog by anti-MUC1-mouse antibodies.

Fluorinated Glycosyl Amino Acids for Mucin‐Like Glycopeptide Antigen Analogues

The aberrant glycosylation profiles of mucin glycoproteins on epithelial tumour cells represent attractive target structures for the development of immunotherapy against cancer. Mucin-type glycopeptides have been successfully investigated as molecularly defined vaccine prototypes for triggering humoral immunity but are susceptible to rapid in vivo degradation. As a potential means to enhance the bioavailabilities of the antigenic structures, hydrolysis-resistant carbohydrate analogues with fluorine substituents at positions C6, C2 and C6 were synthesised and incorporated into the tandem repeat sequence of the mucin MUC1. The resulting pseudo-glycopeptides can be used to elucidate the effects of chemically modified antibody determinants on metabolic and immunological properties.

Multipolar Interactions in the D Pocket of Thrombin: Large Differences between Tricyclic Imide and Lactam Inhibitors.

Two series of tricyclic inhibitors of the serine protease thrombin, imides (+/-)-1-(+/-)-8 and lactams (+/-)-9-(+/-)-13, were analysed to evaluate contributions of orthogonal multipolar interactions with the backbone C=O moiety of Asn98 to the free enthalpy of protein-ligand complexation. The lactam derivatives are much more potent and more selective inhibitors (K(i) values between 0.065 and 0.005 microM, selectivity for thrombin over trypsin between 361- and 1609-fold) than the imide compounds (Ki values between 0.057 and 23.7 microM, selectivity for thrombin over trypsin between 3- and 67-fold). The increase in potency and selectivity is explained by the favorable occupancy of the P-pocket of thrombin by the additional isopropyl substituent in the lactam derivatives. The nature of the substituent on the benzyl ring filling the D pocket strongly influences binding potency in the imide series, with Ki values increasing in the sequence: F < OCH2O < Cl < H < OMe < OH < N(pyr)<< Br. This sequence can be explained by both steric fit and the occurrence of orthogonal multipolar interactions with the backbone C[double bond, length as m-dash]O moiety of Asn98. In contrast, the substituent on the benzyl ring hardly affects the ligand potency in the lactam series. This discrepancy was clarified by the comparison of X-ray structures solved for co-crystals of thrombin with imide and lactam ligands. Whereas the benzyl substituents in the imide inhibitors are sufficiently close (< or =3.5 Angstroms) to the C=O group of Asn98 to allow for attractive orthogonal multipolar interactions, the distances in the lactam series are too large (> or =4 Angstroms) for attractive dipolar contacts to be effective.

Synthesis and biological evaluation of a novel MUC1 glycopeptide conjugate vaccine candidate comprising a 4’-deoxy-4’-fluoro-Thomsen–Friedenreich epitope

Summary The development of selective anticancer vaccines that provide enhanced protection against tumor recurrence and metastasis has been the subject of intense research in the scientific community. The tumor-associated glycoprotein MUC1 represents a well-established target for cancer immunotherapy and has been used for the construction of various synthetic vaccine candidates. However, many of these vaccine prototypes suffer from an inherent low immunogenicity and are susceptible to rapid in vivo degradation. To overcome these drawbacks, novel fluorinated MUC1 glycopeptide-BSA/TTox conjugate vaccines have been prepared. Immunization of mice with the 4’F-TF-MUC1-TTox conjugate resulted in strong immune responses overriding the natural tolerance against MUC1 and producing selective IgG antibodies that are cross-reactive with native MUC1 epitopes on MCF-7 human cancer cells.

Mapping the Fluorophilicity of a Hydrophobic Pocket: Synthesis and Biological Evaluation of Tricyclic Thrombin Inhibitors Directing Fluorinated Alkyl Groups Into the P Pocket

In the completion of our fluorine scan of tricyclic inhibitors to map the fluorophilicity/fluorophobicity of the thrombin active site, a series of 11 new ligands featuring alkyl, alkenyl, and fluoroalkyl groups was prepared to explore fluorine effects on binding into the hydrophobic proximal (P) pocket, lined by Tyru200960A and Trpu200960D, Hisu200957, and Leuu200999. The synthesis of the tricyclic scaffolds was based on the 1,3‐dipolar cycloaddition of azomethine ylides, derived from L‐proline and 4‐bromobenzaldehyde, with N‐(4‐fluorobenzyl)maleimide. Introduction of alkyl, alkenyl, and partially fluorinated alkyl residues was achieved upon substitution of a sulfonyl group by mixed Mg/Zn organometallics followed by oxidation/deoxyfluorination, as well as oxidation/reduction/deoxyfluorination sequences. In contrast, the incorporation of perfluoroalkyl groups required a stereoselective nucleophilic addition reaction at the “upper” carbonyl group of the tricycles, thereby yielding scaffolds with an additional OH, F, or OMe group, respectively. All newly prepared inhibitors showed potent biological activity, with inhibitory constants (Ki values) in the range of 0.008–0.163u2005μM. The X‐ray crystal structure of a protein–ligand complex revealed the exact positioning of a difluoromethyl substituent in the tight P pocket. Fluorophilic characteristics are attributed to this hydrophobic pocket, although the potency of the inhibitors was found to be modulated by steric rather than electronic factors.

Langmuir-Blodgett films of fluorinated glycolipids and polymerizable lipids and their phase separating behavior.

This paper describes the phase separating behavior of Langmuir monolayers from mixtures of different lipids that (i) either carry already a glycopeptide recognition site or can be easily modified to carry one and (ii) polymerizable lipids. To ensure demixing during compression, we used fluorinated lipids for the biological headgroups and hydrocarbon based lipids as polymerizable lipids. As a representative for a lipid monomer, which can be polymerized in the hydrophilic headgroup, a methacrylic monomer was used. As a monomer, which can be polymerized in the hydrophobic tail, a lipid with a diacetylene unit was used (pentacosadiynoic acid, PDA). The fluorinated lipids were on the one hand a perfluorinated lipid with three chains and on the other hand a partially fluorinated lipid with a T(N)-antigen headgroup. The macroscopic phase separation was observed by Brewster angle microscopy, whereas the phase separation on the nanoscale level was observed by atomic force microscopy. It turned out that all lipid mixtures showed (at least) a partial miscibility of the hydrocarbon compounds in the fluorinated compounds. This is positive for pattern formation, as it allows the formation of small demixed 2D patterned structures during crystallization from the homogeneous phase. For miscibility especially a liquid analogue phase proved to be advantageous. As lipid 3 with three fluorinated lipid chains (very stable monolayer) is miscible with the polymerizable lipids 1 and 2, it was mostly used for further investigations. For all three lipid mixtures, a phase separation on both the micrometer and the nanometer level was observed. The size of the crystalline domains could be controlled not only by varying the surface pressure but also by varying the molar composition of the mixtures. Furthermore, we showed that the binary mixture can be stabilized via UV polymerization. After polymerization and subsequent expansion of the barriers, the locked-in polymerized structures are stable even at low surface pressures (10 mN/m), where the unpolymerized mixture did not show any segregation.

Synthesis of Fluorinated Glycosyl Amino Acid Building Blocks for MUC1 Cancer Vaccine Candidates by Microreactor-Assisted Glycosylation

MUC1-type glycopeptides have already shown their potential as possible cancer vaccine candidates. In addition, first examples of fluorinated antigen structures, especially containing the Thomsen-Friedenreich antigen, with similar antibody recognition have been reported. Using microreactor techniques for improvement of the crucial step, the complex glycosylation reactions, is an efficient way to find optimized reaction parameter as well as to circumvent well-known scale-up drawbacks. Besides, this is the first report of continuous flow glycosylations of glycosyl amino acids, in particular with fluorinated glycosyl building blocks.

Patterned monomolecular films from polymerizable and fluorinated lipids for the presentation of glycosylated lipids

This paper deals with pattern formation in Langmuir monolayers of two sets of lipid mixtures that include (1) a fluorinated lipid for phase separation, (2) a polymerizable lipid for stabilization of the patterned structure, and (3) a unit for the presentation of biological recognition units. Differences in the distribution of these functionalities allow a polymerization of dispersed or continuous phase and a placement of the recognition units in crystalline or solid analogue phase. Also, a ternary mixture including a lipid modified with the tandem repeat domain of MUC1 plus a TN-antigen was studied. Based on the biphasic pattern obtained (starlike crystals of up to 50xa0μm with a fine structure of some micrometers), we also verified the potential of the laterally patterned monolayer to stimulate the immune system (quartz crystal microbalance). The second set of lipids combines a highly fluorinated itaconic ester (polymerizable unit) with the natural phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine.