Metabolic Profiling of S-praziquantel: Structure Elucidation Using the Crystalline Sponge Method in Combination with Mass Spectrometry and Nuclear Magnetic Resonance.

Praziquantel (PZQ) is the drug of choice for treatment of the neglected tropical disease schistosomiasis. Although the drug has been extensively used over several decades and its metabolism well studied (several oxidative metabolites are known from literature), the knowledge of the complete structure of some of its metabolites remains elusive. Conventional techniques, such as nuclear magnetic resonance or liquid chromatography mass spectrometry were used in the past to investigate phase I and phase II metabolites of PZQ. These techniques are either limited to provide the complete molecular structure (liquid chromatography mass spectrometry) or require large amount of sample material (NMR), which are not always available when in vitro systems are used for investigation of the metabolites. In this study, we describe new structures of S-PZQ metabolites generated in vitro from human liver microsomes using the crystalline sponge method. After chromatographic separation and purification of the oxidative metabolites, ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry analysis was conducted to narrow down the position of oxidation to a certain part of the molecule. To determine the exact position of hydroxylation, singe-crystal X-ray diffraction analysis of the crystalline sponges and absorbed analyte was used to identify the structure of S-PZQ and its metabolites. The crystalline sponge method allowed for complete structure elucidation of the known metabolites S-trans-4'-hydroxy-PZQ (M1), S-cis-4'-hydroxy-PZQ (M2) and S-/R-11b-hydroxy-PZQ (M6) as well as the unknown metabolites S-9-hydroxy-PZQ (M3) and S-7-hydroxy-S-PZQ (M4). For comparison of structural elucidation techniques, one metabolite (M3) was additionally analyzed using NMR. SIGNIFICANCE STATEMENT: The information content of the metabolic pathway of praziquantel is still limited. The crystalline sponge method allowed the complete structural elucidation of three known and two unknown metabolites of S-praziquantel, using only trace amounts of analyte material, as demonstrated in this study.

Crystalline Sponges as a Sensitive and Fast Method for Metabolite Identification: Application to Gemfibrozil and its Phase I and II Metabolites.

Understanding the metabolism of new drug candidates is important during drug discovery and development, as circulating metabolites may contribute to efficacy or cause safety issues. In the early phase of drug discovery, human in vitro systems are used to investigate human relevant metabolism. Though conventional techniques are limited in their ability to provide complete molecular structures of metabolites (liquid chromatography mass spectrometry) or require a larger amount of material not available from in vitro incubation (nuclear magnetic resonance), we here report for the first time the use of the crystalline sponge method to identify phase I and phase II metabolites generated from in vitro liver microsomes or S9 fractions. Gemfibrozil was used as a test compound. Metabolites generated from incubation with microsomes or S9 fractions, were fractionated using online fraction collection. After chromatographic purification and fractionation of the generated metabolites, single crystal X-ray diffraction of crystalline sponges was used to identify the structure of gemfibrozil metabolites. This technique allowed for complete structure elucidation of 5'-CH2OH gemfibrozil (M1), 4'-OH gemfibrozil (M2), 5'-COOH gemfibrozil (M3), and the acyl glucuronide of gemfibrozil, 1-O-ß-glucuronide (M4), the first acyl glucuronide available in the Cambridge Crystallographic Data Centre. Our study shows that when optimal soaking is possible, crystalline sponges technology is a sensitive (nanogram amount) and fast (few days) method that can be applied early in drug discovery to identify the structure of pure metabolites from in vitro incubations. SIGNIFICANCE STATEMENT: Complete structure elucidation of human metabolites plays a critical role in early drug discovery. Low amounts of material (nanogram) are only available at this stage and insufficient for nuclear magnetic resonance analysis. The crystalline sponge method has the potential to close this gap, as demonstrated in this study.

X-ray Structure Analysis of N-Containing Nucleophilic Compounds by the Crystalline Sponge Method.

Crystalline sponges (CS) used under a set of standard conditions have often failed to give viable N-containing nucleophilic compounds. Despite the high affinity of these nucleophiles to the binding sites of the CS, N-containing compounds considerably harm the coordination framework of the CS during the guest-soaking step. Herein, it is disclosed that these compounds are efficiently absorbed into the CS, without harming the host, under mild conditions (<4 °C, <2 µg) that normally do not work for common organic guests. Moreover, the use of ZnCl2 as the metal component of CS significantly improved the tolerance and robustness of the host framework toward N-containing compounds. Out of 22 drug (or drug-like) N-containing compounds chosen from the WHO model list of essential medicines, we succeeded in analyzing 17 analytes with the modified protocols and/or by using the ZnCl2 -noded CS. This demonstrates that the CS method is now a practical tool for drug-discovery research in pharmaceutical industries.

Brønsted instead of Lewis acidity in functionalized MIL-101Cr MOFs for efficient heterogeneous (nano-MOF) catalysis in the condensation reaction of aldehydes with alcohols.

Porous chromium(III) 2-nitro-, 2-amino-, and nonfunctionalized terephthalate (MIL-101Cr) metal organic frameworks are heterogeneous catalysts for diacetal formation from benzaldehyde and methanol (B-M reaction) as well as other aldehydes and alcohols. MIL-101Cr-NO2 obtained by direct reaction between CrO3 and 2-nitro-terephthalate showed the highest activity with 99% conversion in the B-M reaction in 90 min and turnover numbers of 114. The activity decreased in the order MIL-101Cr-NO2 > MIL-101Cr > MIL-101Cr-NH2. Within different samples of nonfunctionalized MIL-101Cr the activity increased with surface area. Methanol gas sorption of the different MIL materials correlates with the BET surface area and pore volume but not with the diacetalization activity. Benzaldehyde adsorption from heptane showed no significant difference for the different MILs. Gas sorption studies of CD3CN to probe for a higher Lewis acidity in MIL-101Cr-NO2 remained inconclusive. A high B-M catalytic activity of wet MIL-101Cr-NO2 excluded significant contributions from coordinatively unsaturated Lewis-acid sites. pH measurements of methanol dispersions of the MIL materials gave the most acidic pH (as low as 1.9) for MIL-101Cr-NO2, which significantly increased over MIL-101Cr (3.0) to MIL-101Cr-NH2 (3.3). The increase in acidity is of short range or a surface effect to the heterogeneous MIL particles as protons dissociating from the polarized aqua ligands (Cr-OH2) have to stay near the insoluble counteranionic framework. The variation in Brønsted acidity of MIL-101Cr-NO2 > MIL-101Cr ≈ MIL-101Cr-NH2 correlates with the withdrawing effect of NO2 and the diacetalization activity. The catalytic B-M activity of soluble, substitution-inert, and acidic Cr(NO3)3·9H2O supports the Brønsted-acid effect of the MIL materials. Filtration and centrifugation experiments with MIL-101Cr-NO2 revealed that about 2/3 of the catalytic activity comes from nano-MOF particles with a diameter below 200 nm. The MIL-101Cr-NO2 catalysts can be recycled five times with very little loss in activity. The diacetalization activity of MIL-101Cr-NO2 decreases with the alcohol chain length from methanol over ethanol, n-propanol, n-butanol, to almost inactive n-pentanol, while conversions for benzaldehyde, paratolylaldehyde, 4-chlorobenzaldehyde, and cyclohexanone all reach 90% or more after 90 min.

"Directed" assembly of metallacalix[n]arenes with pyrimidine nucleobase ligands of low symmetry: metallacalix[n]arene derivatives of cis-[a2M(cytosine-N3)2]2+ (M=Pt(II), Pd(II); n=4 and 6).

Pyrimidine (pym) ligands with their two endocyclic N-donor atoms provide 120° angles for molecular constructs, which, with the 90° angle metal fragments cis-a(2)M(II) (M=Pt, Pd; a=NH(3) or a(2)=diamine), form cyclic complexes known as metallacalix[n]arenes (with n=3, 4, 6, 8, …). The number of possible isomers of these species depends on the symmetry of the pym ligand. Although highly symmetrical (C(2v)) pym ligands form a single linkage isomer for any n and can adopt different conformations (e.g., cone, partial cone, 1,3-alternate, and 1,2-alternate in the case of n=4), low-symmetry pym ligands (C(s)) can produce a higher number of linkage isomers (e.g., four in the case of n=4) and a large number of different conformers. In the absence of any self-sorting bias, the number of possible species derived from a self-assembly process between cis-a(2)M(II) and a C(s)-symmetrical pym ligand can thus be very high. By using the C(s)-symmetric pym nucleobase cytosine, we have demonstrated that the number of feasible isomers for n=4 can be reduced to one by applying preformed building blocks such as cis-[a(2)M(cytosine-N3)(2)](n+) or cis-[a(2)M(cytosinate-N1)(2)] (for the latter, see the accompanying paper: A. Khutia, P. J. Sanz Miguel, B. Lippert, Chem. Eur. J. 2011, 17, DOI: 10.1002/chem.2010002723) and treating them with additional cis-a(2)M(II) . Moreover, intramolecular hydrogen-bonding interactions between the O2 and N4H(2) sites of the cytosine ligands reduce the number of possible rotamers to one. This approach of the "directed" assembly of a defined metallacalix[4]arene is demonstrated.

"Directed" assembly of metallacalix[n]arenes with pyrimidine nucleobase ligands of low symmetry: interchanging metals in mixed-metal metallacalix[4]arenes and incorporating additional metals at the exocyclic groups.

The pyrimidine (pym) nucleobase cytosine (H(2)C) forms cyclic ring structures ("metallacalix[n]arenes") when treated with square-planar cis-a(2)M(II) entities (M=Pt, Pd; a=NH(3) or a(2)=diamine). The number of possible linkage isomers for a given n and the number of possible rotamers can be substantially reduced if a "directed" approach is pursued. Hence, two cytosine ligands are bonded in a defined way to a kinetically robust platinum corner stone. In the accompanying paper (Part I: A. Khutia, P. J. Sanz Miguel, B. Lippert, Chem. Eur. J. 2010, 17, DOI: 10.1002/chem.2010002722) we have demonstrated this principle by allowing cis-[Pta(2)(H(2)C-N3)(2)](2+) to react with (en)Pd(II) to give cycles of (N1,N3⋅N3,N1▪)(x) (with x=2 or 3; ⋅ represents Pt(II) and ▪ represents Pd(II)). In an extension of this work we have now prepared cis-[Pta(2)(HC-N1)(2)] (1; HC=monoanion of cytosine) and treated it with (bpy)Pd(II) (bpy=2,2'-bipyridine) to give the Pt(2) Pd(2) cycle cis-[{Pt(NH(3))(2)(N1-HC-N3)(2)Pd(bpy)}(2)](NO(3))(4) ⋅13H(2)O (5) with the coordination sites of the metals inverted; hence, platinum is bonded to N1 and palladium is bonded to N3 sites. Again, not only the expected single linkage isomer is formed, but at the same time the solid-state structure and (1)H NMR spectroscopy reveal the preferential occurrence of a single rotamer (1,3-alternate). The addition of (bpy)Pd(II) to 5 led to the formation of Pd(6) Pt(2) complex 6 in which the exocyclic N4H(2) groups of the cytosine ligands have undergone deprotonation and chelate four more (bpy)Pd(II) entities through the O2 and N4H sites. With a large excess of (bpy)Pd(II) over 5 (4:1), cis-(NH(3))(2) Pt(II) is eventually substituted by (bpy)Pd(II) to give the Pd(8) complex 7. In both 6 and 7 stacks of three (bpy)Pd(II) entities occur. The linkage isomer of 5,cis-[{Pt(NH(3))(2)(N3-HC-N1)(2)Pd(bpy)}(2)](NO(3))(4) ⋅9H(2)O (8), has been structurally characterized and the two complexes compared. The acid/base properties of cis-[Pt(NH(3))(2)(H(2)C-N1)(2)] (1) have been determined and compared with those of the corresponding N3 isomer. The complexation of AgCl by 1 is reported.

Crystalline sponge affinity screening: A fast tool for soaking condition optimization without the need of X-ray diffraction analysis.

Structural elucidation of small molecules only available in low quantity (nanogram) is one of the big advantages of the crystalline sponge method. The optimization of various soaking parameters is crucial for effective analyte absorption and repetitive positioning in the pores of the crystal. Time-consuming X-ray diffraction measurements are necessary for data collection and confirmation of successful guest inclusion. In this work, we report a screening method to select optimal soaking conditions without the need of single-crystal X-ray diffraction analysis for individual compounds and mixtures. 14 substances were chosen as test compounds. Parallel guest soaking of individual compounds and mixtures was conducted using various soaking conditions. After evaporation of solvent, excessive material was removed, and guest molecules released through dissolution of the framework. Liquid chromatography-tandem mass spectrometry allowed the estimation of analyte trapped in the pores and the selection of optimal soaking condition dependent on the highest amount of analyte to crystal size (affinity factor). The tool allowed subsequent crystallographic analysis of ten compounds with minimal experiment time. Additionally, a study to examine the lower limit of detection of the crystalline sponge method was conducted. Determination of two target analytes was possible using only 5 ng of sample. Our study shows the potential of an affinity screening to prioritize soaking parameters by estimation of the guest concentration in a single crystal for one or multiple target compounds within a short period of time.

Isomerism with metallacalix[4]arenes of the nonsymmetrical pyrimidine nucleobase cytosine: how connectivity and rotamer state determine the topology of multinuclear derivatives.

Two cyclic octanuclear complexes, 2 and 3, of cation composition [{Pd(bpy)}(8)C(4)](8+) (bpy = 2,2'-bipyridine) form side by side when [Pd(bpy)(H(2)O)(2)](2+) and cytosine (H(2)C) are reacted in water. The two complexes are isomers, composed of central metallacalix[4]arene backbones to which four additional Pd(bpy) units are bonded pairwise to exocyclic groups of the C(2-) ligands. As a consequence of differences in the N1-N3 connectivity patterns of the two central Pd(4)C(4) rings and 1,3-alternate rotamer states of cytosinate in both compounds, the spatial arrangements of exocyclic groups are distinctly different, leading to two Pd(3) stacks and two Pd(1) entities in 2, yet to four Pd(2) stacks in 3.

Molecular Architectures Derived from Metal Ions and the Flexible 3,3'-Bipyridine Ligand: Unexpected Dimer with Hg(II).

The flexible ditopic ligand 3,3'-bipyridine (3,3'-bpy) has been reacted with a series of transition metal species (Ag(+), Hg(2+), cis-a(2)M(2+) (a = NH(3) or a(2) = en; M = Pt, Pd), trans-a(2)Pt(2+) (a = NH(3))) in an attempt to produce discrete cyclic constructs. While Ag(+) gave a polymeric structure {[Ag(3,3'-bpy)](ClO(4)) . H(2)O}(n) (1), with all other metal entities cyclic structures were formed. Interestingly, Hg(CH(3)COO)(2) produced a dinuclear complex [Hg(3,3'-bpy)(CH(3)COO)(2)](2) . 3H(2)O (2), in which the two 3,3'-bpy ligands adopt a cis-orientation of the coordinating pyridyl entities. With cis-(NH(3))(2)Pt(2+), a cyclic complex 4 was isolated in crystalline form which, according to HRMS, is a trimer. With trans-(NH(3))(2)Pt(2+), different species are formed according to (1)H NMR spectroscopy, the nature of which was not established.

Experimental and computational studies of selective recognition of Hg2+ by amide linked lower rim 1,3-dibenzimidazole derivative of calix[4]arene: species characterization in solution and that in the isolated complex, including the delineation of the nanostructures.

Amide linked lower rim 1,3-dibenzimidazole derivative of calix[4]arene, L has been shown to be sensitive and selective to Hg(2+) in aqueous acetonitrile solution based on fluorescence spectroscopy, and the stoichiometry of the complexed species has been found to be 1:1. The selectivity of L toward Hg(2+) has been shown among 11 M(2+) ions, viz., Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), Ca(2+), and Mg(2+) studied, including those of the mercury group and none of these ions impede the recognition of Hg(2+) by L. Role of the solvent on the recognition of Hg(2+) has been demonstrated. The role of calix[4]arene platform and the benzimidazole moieties in the recognition of Hg(2+) by L has been delineated upon performing such studies with five different molecules of relevance as reference molecular systems. The binding cores formed by the receptor L and the reference compounds have been established based on the single crystal XRD structures, and the preferential metal ion binding cores have been discussed. The binding of Hg(2+) with L has been further established based on (1)H and (13)C NMR, ESI MS, absorption, and fluorescence lifetime measurements. Some of these techniques have been used to establish the stoichiometry of the species formed. The complex species formed between L and Hg(2+) have been isolated and characterized and found to be 1:1 species even in the isolated complex. Whereas transmission electron microscopy (TEM), atomic force microscopy (AFM), and scanning electron microscopy (SEM) provided the nanostructural behavior of L, the TEM and SEM demonstrated that the mercury complex has different characteristics when compared to L. The TEM, SEM, and powder XRD studies revealed that whereas L is crystalline, that of the mercury complex is not, perhaps a reason for not being able to obtain single crystals of the complex. Binding characteristics of Hg(2+) toward L have been established based on the DFT computational calculations.

The crystalline sponge method updated.

Crystalline sponges are porous metal complexes that can absorb and orient common organic molecules in their pores and make them observable by conventional X-ray structure analysis (crystalline sponge method). In this study, all of the steps in the crystalline sponge method, including sponge crystal preparation, pore-solvent exchange, guest soaking, data collection and crystallographic analysis, are carefully examined and thoroughly optimized to provide reliable and meaningful chemical information as chemical crystallography. Major improvements in the method have been made in the guest-soaking and data-collection steps. In the soaking step, obtaining a high site occupancy of the guest is particularly important, and dominant parameters for guest soaking (e.g. temperature, time, concentration, solvents) therefore have to be optimized for every sample compound. When standard conditions do not work, a high-throughput method is useful for efficiently optimizing the soaking conditions. The X-ray experiments are also carefully re-examined. Significant improvement of the guest data quality is achieved by complete data collection at high angle regions. The appropriate disorder treatment of the most flexible ZnI2 portions of the host framework and refinement of the solvents filling the remaining void are also particularly important for obtaining better data quality. A benchmark test for the crystalline sponge method toward an achiral molecule is proposed with a guaiazulene guest, in which the guest structure (with ∼ 100% site occupancy) is refined without applying any restraints or constraints. The obtained data quality with R int = 0.0279 and R 1 = 0.0379 is comparable with that of current conventional crystallographic analysis for small molecules. Another benchmark test for this method toward a chiral molecule is also proposed with a santonin guest. The crystallographic data obtained [R int = 0.0421, R 1 = 0.0312, Flack (Parsons) = -0.0071 (11)] represents the potential ability of this method for reliable absolute structure determination.

Programming MIL-101Cr for selective and enhanced CO2 adsorption at low pressure by postsynthetic amine functionalization.

MIL-101Cr fully or partially (p) postsynthetically modified with nitro (-NO2) or amino (-NH2) groups was shown to be a robust, water stable, selective and enhanced carbon dioxide (CO2) adsorption material with the amine-functionality. The highly microporous amine-modified frameworks (up to 1.6 cm(3) g(-1) total pore volume) exhibit excellent thermal stability (>300 °C) with BET surface areas up to 2680 m(2) g(-1). At 1 bar (at 273 K) the gases CO2, CH4 and N2 are adsorbed up to 22.2 wt%, 1.67 wt% and 2.27 wt%, respectively. The two amine-modified MIL-101Cr-NH2 (4) and MIL-101Cr-pNH2 (5) showed the highest gas uptake capacities in the series with high ratios for the CO2 : N2 and CO2 : CH4 selectivities (up to 119 : 1 and 75 : 1, respectively, at 273 K). Comparison with non-modified MIL-101Cr traces the favorable CO2 adsorption properties of MIL-101Cr-NH2 (4) and MIL-101Cr-pNH2 (5) to the presence of the Lewis-basic amine groups. MIL-101Cr-NH2 (4) has a high isosteric heat of adsorption of 43 kJ mol(-1) at zero surface coverage and also >23 kJ mol(-1) over the entire adsorption range, which is well above the heat of liquefaction of bulk CO2. Large CO2 uptake capacities of amine-functionalized 4 and 5, coupled with high adsorption enthalpy, high selectivities and proven long-term water stability, make them suitable candidates for capturing CO2 at low pressure from gas mixtures including the use as a CO2 sorbent from moist air.

Hybrids between classical and metallacalix[4]arenes based on uracil and cis-Pt(II)L2 entities (L = P(Ph)3 or L2 = 2,2'-bipyridine).

The combination of bis(1-methyluracil-5-yl)methane with square-planar Pt(II) entities yields cyclic compounds with the heterocyclic uracil rings alternatingly linked by methylene groups and Pt(II) ions.