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 CF, COH, and COMe 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 log D. The biological assays showed that all compounds are thrombin inhibitors with activities between Ki=0.08 and 2.17 μM. 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 N atom of His 57 which is hydrogen bonded to the catalytic Ser 195. However, for energetically similar polar interactions of CF, COH, and COMe to occur, sufficient space is necessary for the accommodation of the Me group of the COMe residue, and a H‐bond acceptor must be present to prevent unfavorable desolvation of the COH residue.

[1]  F. Diederich,et al.  Design of Novel, Nonpeptidic Thrombin Inhibitors and Structure of a Thrombin–Inhibitor Complex , 1995 .

[2]  Robert Huber,et al.  The refined 1.9 A crystal structure of human alpha‐thrombin: interaction with D‐Phe‐Pro‐Arg chloromethylketone and significance of the Tyr‐Pro‐Pro‐Trp insertion segment. , 1989 .

[3]  Bruce E. Smart,et al.  Fluorine substituent effects (on bioactivity) , 2001 .

[4]  J. Janc,et al.  Engineering inhibitors highly selective for the S1 sites of Ser190 trypsin-like serine protease drug targets. , 2002, Chemistry & biology.

[5]  L. M. Lima,et al.  Bioisosterism: a useful strategy for molecular modification and drug design. , 2005, Current medicinal chemistry.

[6]  P. Kirsch.  Modern Fluoroorganic Chemistry: Synthesis, Reactivity, Applications , 2004 .

[7]  François Diederich,et al.  Orthogonal multipolar interactions in structural chemistry and biology. , 2005, Angewandte Chemie.

[8]  Chu-Young Kim,et al.  Contribution of Fluorine to Protein−Ligand Affinity in the Binding of Fluoroaromatic Inhibitors to Carbonic Anhydrase II , 2000 .

[9]  D W Banner,et al.  Molecular recognition at the thrombin active site: structure-based design and synthesis of potent and selective thrombin inhibitors and the X-ray crystal structures of two thrombin-inhibitor complexes. , 1997, Chemistry & biology.

[10]  T. Hiyama,et al.  Modern synthetic methods for fluorine-substituted target molecules. , 2004, Angewandte Chemie.

[11]  L. Weber,et al.  DESIGN NEUARTIGER, NICHTPEPTIDISCHER THROMBIN-INHIBITOREN UND STRUKTUR EINES THROMBIN: INHIBITOR-KOMPLEXES , 1995 .

[12]  D. Banner,et al.  Crystallographic analysis at 3.0-A resolution of the binding to human thrombin of four active site-directed inhibitors. , 1994, The Journal of biological chemistry.

[13]  F. Diederich,et al.  Structure‐Based Design of Nonpeptidic Thrombin Inhibitors: Exploring the D‐Pocket and the Oxyanion Hole , 2002 .

[14]  D. Hoekman.  Exploring QSAR Fundamentals and Applications in Chemistry and Biology, Volume 1. Hydrophobic, Electronic and Steric Constants, Volume 2 J. Am. Chem. Soc. 1995, 117, 9782 , 1996 .

[15]  W. R. Dolbier,et al.  Fluorine chemistry at the millennium , 2005 .

[16]  Masaki Shimizu,et al.  Moderne Synthesemethoden für fluorierte Verbindungen , 2005 .

[17]  O. Mitsunobu.  The Use of Diethyl Azodicarboxylate and Triphenylphosphine in Synthesis and Transformation of Natural Products , 1981 .

[18]  B. Smart,et al.  Organofluorine chemistry : principles and commercial applications , 1994 .

[19]  E. LaVoie,et al.  Bioisosterism: A Rational Approach in Drug Design. , 1996, Chemical reviews.

[20]  Albert J. Leo,et al.  Calculating log P(oct) with no missing fragments; The problem of estimating new interaction parameters , 2000 .

[21]  Manfred Kansy,et al.  Fluorine Interactions at the Thrombin Active Site: Protein Backbone Fragments HCαCO Comprise a Favorable CF Environment and Interactions of CF with Electrophiles , 2004, Chembiochem : a European journal of chemical biology.

[22]  Paul R. Gerber,et al.  MAB, a generally applicable molecular force field for structure modelling in medicinal chemistry , 1995, J. Comput. Aided Mol. Des..

[23]  M. Schlosser.  Parametrisierung von Substituenten – der Einfluß von Fluor und anderen Heteroatomen auf OH-, NH- und CH-Aciditäten , 1998 .

[24]  Thomas B. Tschopp,et al.  Enantiomerically Pure Thrombin Inhibitors for Exploring the Molecular-Recognition Features of the Oxyanion Hole , 2004 .

[25]  M. Schlosser.  Parametrization of Substituents: Effects of Fluorine and Other Heteroatoms on OH, NH, and CH Acidities. , 1998, Angewandte Chemie.

[26]  Erik Verner,et al.  Exploiting subsite S1 of trypsin-like serine proteases for selectivity: potent and selective inhibitors of urokinase-type plasminogen activator. , 2001, Journal of medicinal chemistry.

[27]  R. Schinazi,et al.  A COMPLETELY DIASTEREOSELECTIVE ELECTROPHILIC FLUORINATION OF A CHIRAL, NONCARBOHYDRATE SUGAR RING PRECURSOR : APPLICATION TO THE SYNTHESIS OF SEVERAL NOVEL 2'-FLUORONUCLEOSIDES , 1998 .

[28]  F. Diederich,et al.  Orthogonale multipolare Wechselwirkungen in chemischen und biologischen Strukturen , 2005 .

[29]  Paul Seiler,et al.  A fluorine scan of thrombin inhibitors to map the fluorophilicity/fluorophobicity of an enzyme active site: evidence for C-F...C=O interactions. , 2003, Angewandte Chemie.

[30]  F. Diederich,et al.  Synthesis of Novel Nonpeptidic Thrombin Inhibitors , 2000 .

[31]  J. Hall,et al.  The action of thrombin on peptide p-nitroanilide substrates. Substrate selectivity and examination of hydrolysis under different reaction conditions. , 1983, Biochimica et biophysica acta.

[32]  Corwin Hansch,et al.  Role of hydrophobic effects in mechanistic QSAR , 1999 .

[33]  M. Schlosser,et al.  Alpha-fluorinated ethers, thioethers, and amines: anomerically biased species. , 2005, Chemical reviews.

[34]  Some influences of fluorine in bioorganic chemistry , 1997 .

[35]  Y. Itoh,et al.  Fluorinated carbonyl and olefinic compounds: basic character and asymmetric catalytic reactions. , 2004, Chemical reviews.

[36]  N. Kitteringham,et al.  Metabolism of fluorine-containing drugs. , 2001, Annual review of pharmacology and toxicology.

[37]  Martin Stahl,et al.  Fluorine in Medicinal Chemistry , 2004, Chembiochem : a European journal of chemical biology.

[38]  F. Diederich,et al.  A fluorine scan of the phenylamidinium needle of tricyclic thrombin inhibitors: effects of fluorine substitution on pKa and binding affinity and evidence for intermolecular C-F...CN interactions. , 2004, Organic & biomolecular chemistry.

[39]  P. Jeschke.  The Unique Role of Fluorine in the Design of Active Ingredients for Modern Crop Protection , 2004, Chembiochem : a European journal of chemical biology.

[40]  Maria Cristina Burla,et al.  SIR97: a new tool for crystal structure determination and refinement , 1999 .

[41]  S. Withers,et al.  Glycosyl fluorides in enzymatic reactions. , 2000, Carbohydrate research.

[42]  C. Fierke,et al.  Twisted amides inferred from QSAR analysis of hydrophobicity and electronic effects on the affinity of fluoroaromatic inhibitors of carbonic anhydrase. , 2002, The Journal of organic chemistry.

[43]  K. Toshima.  Glycosyl fluorides in glycosidations. , 2000, Carbohydrate research.

[44]  K Gubernator,et al.  Design and synthesis of potent and highly selective thrombin inhibitors. , 1994, Journal of medicinal chemistry.

[45]  A. Pinner,et al.  Umwandlung der Nitrile in Imide , 1878 .

[46]  W. Middleton.  New fluorinating reagents. Dialkylaminosulfur fluorides , 1975 .

[47]  F. Diederich,et al.  A weak attractive interaction between organic fluorine and an amide group. , 2004, Angewandte Chemie.