Validation of experimental charge densities: refinement of the macrolide antibiotic roxithromycin.

Multipole refinements of larger organic molecules have so far been limited to a few exceptional cases. We report an investigation of the detailed experimental electron-density distribution (EDD) of roxithromycin, a macrolide antibiotic consisting of 134 atoms. Although the experimental multipole refinement on high-resolution synchrotron data converged smoothly, validation of the electron density by calculation of an `experiment minus invariom' difference density revealed conformational disorder of the H atoms. Hydrogen disorder is shown to affect the EDD, the electrostatic potential and atomic properties as defined by Bader's quantum theory of atoms in molecules. A procedure to obtain the electron density distribution in the presence of disorder is proposed.

Introduction and validation of an invariom database for amino-acid, peptide and protein molecules.

A database of invarioms for structural refinement of amino-acid, oligopeptide and protein molecules is presented. The spherical scattering factors of the independent atom or promolecule model are replaced by ;individual' aspherical scattering factors that take into account the chemical environment of a bonded atom. All amino acids were analysed in terms of their invariom fragments. In order to generate 73 database entries that cover this class of compounds, 37 model compounds were geometry-optimized and theoretical structure factors were calculated. Multipole refinements were then performed on these theoretical structure factors to yield the invariom database. Validation of this database on an extensive number of experimental small-molecule crystal structures of varying quality and resolution shows that invariom modelling improves various figures of merit. Differences in figures of merit between invariom and promolecule models give insight into the importance of disorder for future protein-invariom refinements. The suitability of structural data for application of invarioms can be predicted by Cruickshank's diffraction-component precision index [Cruickshank (1999), Acta Cryst. D55, 583-601].

Atomic and bond topological properties of the tripeptide L-alanyl-L-alanyl-L-alanine based on its experimental charge density obtained at 20 K.

A 20 K high resolution X-ray data set of L-Ala-L-Ala-L-Ala*1/2 H2O was measured using an ultra-low temperature laboratory setup, that combines area detection and a closed cycle helium cryostat. The charge density determination includes integration of atomic basins and topological analysis according to Bader's quantum theory of atoms in molecules. Two tripeptide units are found in the asymmetric unit, allowing the assessment of transferability of bond topological and atomic properties taking also into consideration previous data of oligopeptides. With respect to invariom modeling the limits of such transferability are investigated and the results of this study show the validity of the nearest/next-nearest neighbour approximation and support the use of database approaches for electron density modeling of macromolecules.

Submolecular partitioning of morphine hydrate based on its experimental charge density at 25 K.

The electron density distribution of morphine hydrate has been determined from high-resolution single-crystal X-ray diffraction measurements at 25 K. A topological analysis was applied and, in order to analyze the submolecular transferability based on an experimental electron density, a partitioning of the molecule into atomic regions was carried out, making use of Bader's zero-flux surfaces to yield atomic volumes and charges. The properties obtained were compared with the theoretical calculations of smaller fragment molecules, from which the complete morphine molecule can be reconstructed, and with theoretical studies of another opiate, Oripavine PEO, reported in the literature.

The invariom model and its application: refinement of D,L-serine at different temperatures and resolution.

Three X-ray data sets of the same D,L-serine crystal were measured at temperatures of 298, 100 and 20 K. These data were then evaluated using invarioms and the Hansen & Coppens aspherical-atom model. Multipole populations for invarioms, which are pseudoatoms that remain approximately invariant in an intermolecular transfer, were theoretically predicted using different density functional theorem (DFT) basis sets. The invariom parameters were kept fixed and positional and thermal parameters were refined to compare the fitting against the multi-temperature data at different resolutions. The deconvolution of thermal motion and electron density with respect to data resolution was studied by application of the Hirshfeld test. Above a resolution of sin theta/lambda approximately 0.55 A-1, or d approximately 0.9 A, this test was fulfilled. When the Hirshfeld test is fulfilled, a successful modeling of the aspherical electron density with invarioms is achieved, which was proven by Fourier methods. Molecular geometry improves, especially for H atoms, when using the invariom method compared to the independent-atom model, as a comparison with neutron data shows. Based on this example, the general applicability of the invariom concept to organic molecules is proven and the aspherical density modeling of a larger biomacromolecule is within reach.

Atomic volumes and charges in a system with a strong hydrogen bond: L-tryptophan formic acid.

A high-resolution X-ray diffraction data set was collected within 48 h at 100 K with synchrotron radiation and area detection. A full topological analysis was applied to the resulting electron-density model. This analysis was followed by a Bader partitioning making use of the zero-flux surfaces of the electron-density gradient vector field. The atomic and bonding properties obtained were compared with the results of previous experimental studies, and with theoretical calculations for the title complex and free tryptophan as reported in the literature. The agreement between experiment and theory is similar to the agreement between different theoretical calculations. There is no charge transfer via the strong hydrogen bond; however, its strength is indicated by the very small atomic volume of the H atom involved.

Reproducability and transferability of topological properties; experimental charge density of the hexapeptide cyclo-(D,L-Pro)2-(L-Ala)4 monohydrate.

The charge density of a hexapeptide was determined from high-resolution CCD area-detector experiments at 100 K. Two datasets, one from a rotating anode and a second one from synchrotron radiation, were measured and the results are compared. The data are interpreted in terms of the 'rigid pseudoatom' model. The topology of the experimental density is analyzed and compared with the topology of the constituting amino acids, and shows good agreement. All critical points of the electron density at the covalent and hydrogen bonds, as well as those of the Laplacian, were located. With respect to the transferability of electronic and bond topological properties the six peptide bonds were compared with values given in the literature.

Intra- and intermolecular topological properties of amino acids: a comparative study of experimental and theoretical results.

The charge densities rho(r) of the six amino acids L-Asn.H(2)O, DL-Glu.H(2)O, DL-Lys.HCl, DL-Pro.H(2)O, DL-Ser, and DL-Val were determined from high-resolution X-ray diffraction experiments at 100 K using synchrotron radiation and area detection (CCD) techniques. Bond topological parameters derived from these densities and from those of six additional amino acids published earlier are compared to each other and to the results of ab initio calculations. Experimental and theoretical properties for each chemically equivalent bond are in a fair agreement, and their variances are of similar magnitude. A noticeable outlier is the positive curvature of the density at the bond critical point, for which no correlation between the experimental and theoretical values can be established. The location of nonbonded valence shell charge concentrations derived from the crystalline densities scatter in a wider range than those obtained for the isolated molecules.

Racemic dipeptide glycyl-DL-leucine at 120 K.

The structure of glycyl-DL-leucine, C(8)H(16)N(2)O(3), has been determined at 120 K by single-crystal X-ray diffraction. In addition to three N-H.O-type hydrogen bonds of the positively charged RNH(3)(+) group of the zwitterionic molecule, an intermolecular N-H. O contact exists between the peptide bond and the carboxylate group. Four hydrogen-bond cycles were identified, giving a complex pattern.

DL-arginine monohydrate at 100 K.

In the title compound, C(6)H(14)N(4)O(2).H(2)O, the alpha-amino group is neutral. The molecular side chain including the guanidinium group is not fully extended, having a near gauche-gauche conformation [chi(3) = 59.0(1)degrees; chi(4) = 72.8(1)degrees]. The network of hydrogen bonds stabilizing the crystal lattice includes those formed between the deprotonated and negatively charged alpha-carboxylate groups and the positively charged amino groups of the guanidinium group of neighbouring molecules. N-H...O=C and water-mediated N-H...O hydrogen bonds link individual molecules to produce pairs of spiral motifs laterally connected by N-H...O and C-H...O hydrogen bonds.

Chiral acetals as stereoinductors: diastereoface selective alkylation of dihydrobenzoxazine-derived amide enolates

Novel dihydrobenzoxazine-derived acetals of type 3 have been developed for asymmetric C-alkylations of propionyl amide enolates. High stereoselectivities are obtained for amides 15 and 22 which are rationalized in terms of intramolecular metal chelate formation.

3-(tert-Butyloxycarbonylamino)bicyclo

In the room-temperature X-ray structure of the N-Boc-protected derivative of the novel 3-aminobicyclo[1.1.1]pentanecarboxylic acid, C(11)H(17)NO(4), the interbridgehead distance in the bicyclo[1.1. 1]pentane cage is 1.852 (2) A. The carboxyl and parts of the blocked amino group are almost in plane with one of the cage triangles. N-H. O and O-H.O hydrogen bonds generate infinite corrugated molecular chains in the crystal lattice.

New histamine H(3)-receptor ligands of the proxifan series: imoproxifan and other selective antagonists with high oral in vivo potency.

Histamine H(3)-receptor antagonists of the proxifan series are described. The novel compounds possess a 4-(3-(phenoxy)propyl)-1H-imidazole structure and various functional groups, e.g., an oxime moiety, on the phenyl ring. Synthesis of the novel compounds and X-ray crystallography of one highly potent oxime derivative, named imoproxifan (4-(3-(1H-imidazol-4-yl)propyloxy)phenylethanone oxime), are described. Most of the title compounds possess high antagonist potency in histamine H(3)-receptor assays in vitro as well as in vivo in mouse CNS following po administration. Structure-activity relationships are discussed. Imoproxifan displays subnanomolar potency on a functional assay on synaptosomes of rat cerebral cortex (K(i) = 0.26 nM). In vivo, imoproxifan increases the central N(tau)-methylhistamine level with an ED(50) of 0.034 mg/kg po. A receptor profile on several functional in vitro assays was determined for imoproxifan, demonstrating high selectivity toward the histamine H(3) receptor for this promising candidate for further development.

Topological properties of the peptide bond in glycyl-L-threonine dihydrate based on a fast synchrotron/CCD-diffraction experiment at 100 K.

The charge density of glycyl-L-threonine dihydrate is extracted from a synchrotron data set of 98405 reflections collected at 100 K with a Bruker CCD area detector up to a resolution of d=0.38 A (sintheta/lambda = 1.32 A 1). The data are interpreted in terms of the "rigid pseudoatom" model. The topology of the experimental density is analyzed and compared with the topology obtained experimentally for the constituting amino acids and to that derived from Hartree-Fock calculations for the isolated molecule. All critical points of the electron density at the covalent and hydrogen bonds, as well as those of the Laplacian, were located, thereby deriving quantitative topological data for the peptide and side chain bonds. Bond topological indices in the dipeptide compare well with those of the corresponding bonds in the building amino acids, thus suggesting transferability of electronic properties of atoms and functional groups when these are derived by Bader's partitioning. Discrepancies between theoretical and experimental results could be attributed to crystal field effects.

Crystal and molecular structures of trifluoroacrylonitrile, F2C=CF-CN, and trifluorovinyl isocyanide, F2C=CF-NC, by low-temperature X-ray crystallography and ab initio calculations.

The structures of trifluoroacrylonitrile, F2C=CF-CN, monoclinic, P2(1/n) (no. 14), a = 8.595(4), b = 8.748(1), c = 5.421(1) A, beta = 102.83(2) degrees, Z = 4, and its thermally unstable isomer trifluorovinyl isocyanide, F2C=CF-NC, monoclinic, P2(1/n), a = 8.501(2), b = 8.828(2), c = 5.599(2) A, beta = 101.11(2) degrees, Z = 4 were determined by X-ray crystal structure analysis at 113 and 128 K, respectively, from single crystals grown by partial melting and gradient cooling in small glass capillaries. Selected experimental bond lengths of F2C=CF-CN/F2C=CF-NC are as follows: C=C 1.326(1)/1.304(2), C...N 1.158(1)/1.167(2) A. The C-F bond lengths of the CF2 group are significantly shorter than those of the CF(NC) and CF(CN) units, respectively. The vibrational frequencies and molecular geometries of this cyanide/isocyanide pair were also calculated by ab initio methods for comparison with the experimental results, which were found to be in general agreement.

Guanidine derivatives as combined thromboxane A2 receptor antagonists and synthase inhibitors.

A new series of omega-disubstituted alkenoic acid derivatives derived from samixogrel 5 were designed and synthesized as combined thromboxane A2 receptor antagonists/thromboxane A2 synthase inhibitors with improved solubility and reduced protein binding compared to 5. Hexenoic acid derivatives with a 3-pyridyl group and 3-(2-cyano-3-alkyl-guanidino)phenyl substituent were found to be optimal with regard to this dual mode of action. The most potent compound, E-6-(3-(2-cyano-3-tert-butyl-guanidino)phenyl)-6-(3-pyridyl)hex-5-eno ic acid, "terbogrel" 32 inhibits the thromboxane A2 synthase in human gel-filtered platelets with an IC50 value of 4.0 +/- 0.5 nM (n = 4). Radioligand binding studies with 3H-SQ 29,548 revealed that 32 blocks the thromboxane A2/endoperoxide receptor on washed human platelets with an IC50 of 11 +/- 6 nM (n = 2) and with an IC50 of 38 +/- 1 nM (n = 15) in platelet-rich plasma. Terbogrel inhibits the collagen-induced platelet aggregation in human platelet-rich plasma and whole blood with an IC50 of 310 +/- 18 nM (n = 8) and 52 +/- 20 nM (n = 6), respectively. This was shown to translate into a potent antithrombotic effect in vivo as demonstrated in studies using a model of arterial thrombosis in rabbits (ED50 = 0.19 +/- 0.07 mg/kg; n = 20). Thus, terbogrel is the first compound with a guanidino moiety demonstrating both a potent TXA2 synthase inhibition and a potent TXA2 receptor antagonism and has been selected for further clinical development.

Low-temperature crystallization and structure determination of N-(trifluoromethyl)formamide, N-(2,2,2-trifluoroethyl)formamide and 2,2,2-trifluoroethyl isocyanide.

Crystals of N-(trifluoromethyl)formamide, C(2)H(2)F(3)NO, (I), N-(2,2,2-trifluoroethyl)formamide, C(3)H(4)F(3)NO, (II), and 2,2,2-trifluoroethyl isocyanide, C(3)H(2)F(3)N, (III), were grown in situ on an X-ray diffractometer and analysed by single-crystal X-ray diffraction methods at low temperatures. Crystal data: (I) orthorhombic, P2(1)2(1)2(1), a = 4.547 (2) Å, b = 5.947 (3) Å, c = 14.731 (9) Å, V = 398.3 (4) Å(3), Z = 4, M(r) = 113.05, T = 143 K, D(x) = 1.885 Mg m(-3); (II) monoclinic, P2(1)/n, a = 4.807 (1) Å, b = 16.707 (3) Å, c = 6.708 (1) Å, beta = 109.90 (1) degrees, V = 506.6 (2) Å(3), Z = 4, M(r) = 127.07, T = 141 K, D(x) = 1.666 Mg m(-3); (III) orthorhombic, P2(1)2(1)2(1), a = 5.668 (2) Å, b = 9.266 (3) Å, c = 8.626 (2) Å, V = 453.0 (2) Å(3), Z = 4, M(r) = 109.06, T = 163 K, D(x) = 1.599 Mg m(-3). The results showed that in the crystal both formamides (I) and (II) are exclusively present in the form of the Z isomer, although measurements of solutions of (I) have shown that the E isomer prevails [Lentz et al. (1987). Angew. Chem. 99, 951-953]. In addition ab initio calculations for (I) predicted the E isomer to be the more stable one. In compound (III) the isocyanide group is staggered with respect to the trifluoroethyl group. In the crystal packing of (I) and (II) intermolecular N-H.O hydrogen bonds generate infinite chains. In (I), these chains are linked to form sheets by C-H.O contacts. In the crystal structure of (III) each isocyanide dipole is surrounded by four electronegative F atoms with intermolecular C.F contacts between 3.4 and 3.5 Å.

Repaglinide and related hypoglycemic benzoic acid derivatives.

The structure-activity relationships in two series of hypoglycemic benzoic acid derivatives (5, 6) were investigated. Series 5 resulted from meglitinide (3) when the 2-methoxy was replaced by an alkyleneimino residue. Maximum activity was observed with the cis-3, 5-dimethyl-piperidino (5h) and the octamethyleneimino (5l) residues. Series 6 resulted from the meglitinide analogon 4 bearing an inversed amido function when the 2-methoxy, the 5-fluoro, and the alpha-methyl residue were replaced by a 2-piperidino, a 5-hydrogen, and a larger alpha-alkyl residue, respectively. An alkoxy residue ortho to the carboxy group further increased activity and duration of action in the rat. The most active racemic compound, 6al (R4 = isobutyl; R = ethoxy), turned out to be 12 times more active than the sulfonylurea (SU) glibenclamide (1). Activity was found to reside predominantly in the (S)-enantiomers. Compared with the SUs 1 and 2 (glimepiride), the most active enantiomer, (S)-6al (AG-EE 623 ZW; repaglinide; ED50 = 10 micro/kg po), is 25 and 18 times more active. Repaglinide turned out to be a useful therapeutic for type 2 diabetic patients; approval was granted recently by the FDA and the EMEA. From investigations on the pharmacophoric groups in compounds of type 5 and 6, it was concluded that in addition to the two already known-the acidic group (COOH; SO2NH) and the amidic spacer (CONH; NHCO)-the ortho residue R1 (alkyleneimino; alkoxy; oxo) must be regarded as a third one. A general pharmacophore model suitable for hypoglycemic benzoic acid derivatives, SUs, and sulfonamides is proposed (Figure 6). Furthermore, from superpositions of low-energy conformations (LECs) of 1, 2, and (S)-6al, it was concluded that a common binding conformation (LEC II; Figure 10B) may exist and that differences in binding to the SU receptor and in the mechanism of insulin release between repaglinide and the two SUs may be due to specific hydrophobic differences.

Accurate experimental electronic properties of dl-proline monohydrate obtained within 1 Day

A 1-day x-ray diffraction experiment on dl-proline monohydrate was performed at 100 kelvin with synchrotron radiation and a charge-coupled device area detection technique. The accuracy of the charge density distribution and of the related electronic properties extracted from these data is comparable or even superior to the accuracy obtained from a 6-week experiment on dl-aspartic acid with conventional x-ray diffraction methods. A data acquisition time of 1 day is comparable to the time needed for an ab initio calculation on the isolated molecules. This technique renders larger molecular systems of biological importance accessible to charge density experiments.