Discovery of Prodrug of MRTX1133 as an Oral Therapy for Cancers with KRASG12D Mutation.

Effective oral therapies are urgently required to treat KRASG12D mutant cancers. Therefore, synthesis and screening were performed for 38 prodrugs of MRTX1133 to identify an oral prodrug of MRTX1133, a KRASG12D mutant protein-specific inhibitor. In vitro and in vivo evaluations revealed prodrug 9 as the first orally available KRASG12D inhibitor. Prodrug 9 exhibited improved pharmacokinetic properties for the parent compound in mice and was efficacious in a KRASG12D mutant xenograft mouse tumor model after oral administration.

Inhibition of bacterial growth and galactosyltransferase activity of WbwC by α, ω-bis(3-alkyl-1H-imidazolium)alkane salts: Effect of varying carbon content.

A series of compounds was designed and synthesized having two imidazolium rings separated by a polymethylene spacer and having alkyl substituents on each of the imidazolium rings. The compounds were assayed for their effects on the activity of galactosyltransferase WbwC, and also on the growth of Gram-negative and Gram-positive bacteria, as well as human cells. The inhibition observed on enzyme activities and cell growth was dependent on the total number of carbons in the spacer and the alkyl substituents on the imidazolium rings. These readily synthesized, achiral compounds have potential as antimicrobial and antiseptic agents.

Solid-State 17O NMR Study of Carboxylic Acid Dimers: Simultaneously Accessing Spectral Properties of Low- and High-Energy Tautomers.

We report a solid-state 17O NMR study of several crystalline carboxylic acids. We found that, while each of these compounds forms discrete hydrogen-bonded dimers in the crystal lattice, their solid-state 17O magic-angle spinning (MAS) NMR spectra display quite different features and different temperature dependencies. We showed that experimentally observed 17O NMR spectral behaviors can be explained as being due to thermal averaging between the two tautomers that are produced as a result of concerted double-hydrogen hopping dynamics within each dimer. In general, the two tautomers have different energies due to intramolecular interactions and crystal packing. From an analysis of variable-temperature 17O MAS NMR spectra, energy asymmetry between the two tautomers was experimentally determined for each of the carboxylic acid compounds studied. The same data analysis also offers an opportunity to simultaneously assess 17O NMR parameters in both low- and high-energy tautomers. We concluded that the periodic plane-wave density functional theory (DFT) calculations can produce reliable 17O NMR parameters (chemical shift and quadrupolar coupling tensors) for both tautomers. The same periodic DFT calculations have also produced reasonable energy asymmetry values for the studied carboxylic acid dimers. We have also observed substantial H/D isotope shifts in solid-state 17O NMR.

Differential inhibition of rat and mouse microsome heme oxygenase by derivatives of imidazole and benzimidazole.

Metalloporphyrin heme oxygenase (HO) inhibitors have made an important contribution to elucidating the role of HO in physiological processes. Nevertheless, their off-target effects have drawn substantial criticism, which prompted us to develop non-porphyrin, azole-based inhibitors of HO. These second-generation HO inhibitors were evaluated using spleen and brain microsomes from rats as native sources of HO-1 and HO-2, respectively. Recently, the use of azole-based inhibitors of HO has been extended to other mammalian species and, as a consequence, it will be important to characterize the inhibitors in these species. The goal of this study was to compare the inhibitory profile of imidazole- and benzimidazole-based inhibitors of HO in a breast-cancer-implanted mouse to that of an untreated rat. For spleen and brain microsomes from both species, HO protein expression was determined by Western blotting and concentration-response curves for imidazole- and benzimidazole-derivative inhibition of HO activity were determined using a headspace gas-chromatographic assay. It was found that the effects on HO activity by imidazole and benzimidazole derivatives were different between the 2 species and were not explained by differences in HO expression. Thus, the HO inhibitory profile should be determined for azole derivatives before they are used in mammalian species other than rats.

Solid-State 17 O†NMR Reveals Hydrogen-Bonding Energetics: Not All Low-Barrier Hydrogen Bonds Are Strong.

While NMR and IR spectroscopic signatures and structural characteristics of low-barrier hydrogen bond (LBHB) formation are well documented in the literature, direct measurement of the LBHB energy is difficult. Here, we show that solid-state 17 O NMR spectroscopy can provide unique information about the energy required to break a LBHB. Our solid-state 17 O NMR data show that the HB enthalpy of the O⋅⋅⋅H⋅⋅⋅N LBHB formed in crystalline nicotinic acid is only 7.7±0.5 kcal mol-1 , suggesting that not all LBHBs are particularly strong.

Solid-state 17O NMR study of 2-acylbenzoic acids and warfarin.

We report synthesis and solid-state 17O NMR characterization of four site-specifically 17O-labeled 2-acylbenzoic acids (2-RC(O)C6H4COOH) where R=H and CH3): 2-[3-17O]formylbenzoic acid, 2-[1,2-17O2]formylbenzoic acid, 2-[3-17O]acetylbenzoic acid, and 2-[1,2,3-17O3]acetylbenzoic acid. In the solid state, both 2-formyl- and 2-acetyl-benzoic acids exist as the cyclic phthalide form each containing a five-membered lactone ring and a cyclic hemiacetal/hemiketal group. Static and magic-angle-spinning 17O NMR spectra were recorded at 14.1 and 21.1T for these compounds, from which the 17O chemical shift and nuclear quadrupolar coupling tensors were determined for each oxygen site. These results represent the first time that 17O NMR tensors are fully characterized for lactone, cyclic hemiacetal, and cyclic hemiketal functional groups. We also report solid-state 17O NMR data for the cyclic hemiketal group an anticoagulant drug, warfarin. Experimental 17O NMR tensors in these compounds were compared with computational results obtained with a periodic DFT code BAND.

Proton Probability Distribution in the O···H···O Low-Barrier Hydrogen Bond: A Combined Solid-State NMR and Quantum Chemical Computational Study of Dibenzoylmethane and Curcumin.

We report a combined solid-state (1H, 2H, 13C, 17O) NMR and plane-wave density functional theory (DFT) computational study of the O···H···O low-barrier hydrogen bonds (LBHBs) in two 1,3-diketone compounds: dibenzoylmethane (1) and curcumin (2). In the solid state, both 1 and 2 exist in the cis-keto-enol tautomeric form, each exhibiting an intramolecular LBHB with a short O···O distance (2.435 Å in 1 and 2.455 Å in 2). Whereas numerous experimental (structural and spectroscopic) and computational studies have been reported for the enol isomers of 1,3-diketones, a unified picture about the proton location within an LBHB is still lacking. This work reports for the first time the solid-state 17O NMR data for the O···H···O LBHBs in 1,3-diketones. The central conclusion of this work is that detailed information about the probability density distribution of the proton (nuclear zero-point motion) across an LBHB can be obtained from a combination of solid-state NMR and plane-wave DFT computations (both NMR parameter calculations and ab initio molecular dynamics simulations). We propose that the precise proton probability distribution across an LBHB should provide a common basis on which different and sometimes seemingly contradicting experimental results obtained from complementary techniques, such as X-ray diffraction, neutron diffraction, and solid-state NMR, can be reconciled.

Solid-State 17O NMR of Unstable Acyl-Enzyme Intermediates: A Direct Probe of Hydrogen Bonding Interactions in the Oxyanion Hole of Serine Proteases.

We report preparation, trapping, and solid-state 17O NMR characterization of three unstable acyl-enzyme intermediates (≈ 26 kDa): p-N,N-dimethylamino-[17O]benzoyl-chymotrypsin, trans-o-methoxy-[17O]cinnamoyl-chymotrypsin, and trans-p-methoxy-[17O]cinnamoyl-chymotrypsin. We show that both the 17O chemical shifts and nuclear quadrupolar parameters obtained for these acyl-enzyme intermediates in the solid state are correlated with their deacylation rate constants measured in aqueous solution. With the aid of quantum mechanical calculations, the experimental 17O NMR parameters were interpreted as to reflect the hydrogen bonding interactions between the carbonyl (C═17O) functional group of the acyl moiety and the two NH groups from the protein backbone (Ser195 and Gly193) in the oxyanion hole, a general feature of all serine proteases. Our results further suggest that the 17O chemical shift and quadrupole coupling constant display distinctly different sensitivities toward different aspects of hydrogen bonding, such as hydrogen bond distance and direction. This work demonstrates the utility of 17O as a useful nuclear probe in NMR studies of enzymes.

Solving the 170-Year-Old Mystery About Red-Violet and Blue Transient Intermediates in the Gmelin Reaction.

The Gmelin reaction between nitroprusside and sulfides in aqueous solution is known to produce two transient intermediates with distinct colors: an initial red-violet intermediate that subsequently converts into a blue intermediate. In this work, we use a combination of multinuclear ((17) O, (15) N, (13) C) NMR, UV/Vis, IR spectroscopic techniques and quantum chemical computation to show unequivocally that the red-violet intermediate is [Fe(CN)5 N(O)S](4-) and the blue intermediate is [Fe(CN)5 N(O)SS)](4-) . While the formation of [Fe(CN)5 N(O)S](4-) has long been postulated in the literature, this study provides the most direct proof of its structure. In contrast, [Fe(CN)5 N(O)SS)](4-) represents the first example of any metal coordination complex containing a perthionitro ligand. The new reaction pathways found in this study not only provide clues for the mode of action of nitroprusside for its pharmacological activity, but also have broader implications to the biological role of H2 S, potential reactions between H2 S and nitric oxide donor compounds, and the possible biological function of polysulfides.

Solid-State (17)O NMR of Oxygen-Nitrogen Singly Bonded Compounds: Hydroxylammonium Chloride and Sodium Trioxodinitrate (Angeli's Salt).

We report a solid-state NMR study of (17)O-labeled hydroxylammonium chloride ([H(17)O-NH3]Cl) and sodium trioxodinitrate monohydrate (Na2[(17)ONNO2]·H2O, Angeli's salt). The common feature in these two compounds is that they both contain oxygen atoms that are singly bonded to nitrogen. For this class of oxygen-containing functional groups, there is very limited solid-state (17)O NMR data in the literature. In this work, we experimentally measured the (17)O chemical shift and quadrupolar coupling tensors. With the aid of plane-wave DFT computation, the (17)O NMR tensor orientations were determined in the molecular frame of reference. We found that the characteristic feature of an O-N single bond is that the (17)O nucleus exhibits a large quadrupolar coupling constant (13-15 MHz) but a rather small chemical shift anisotropy (100-250 ppm), in sharp contrast with the nitroso (O═N) functional group for which both quantities are very large (e.g., 16 MHz and 3000 ppm, respectively).

Structure-Activity Relationships of 1,2-Disubstituted Benzimidazoles: Selective Inhibition of Heme Oxygenase-2 Activity.

Devising ways to up- or down-regulate heme oxygenase activity is attracting much interest as a strategy for the treatment of a variety of disorders. With a view of obtaining compounds that exhibit high potency and selectivity as inhibitors of the heme oxygenase-2 (HO-2) isozyme (constitutive) relative to the heme oxygenase-1 (HO-1) isozyme (inducible), several 1,2-disubstituted 1H-benzimidazoles were designed and synthesized. Specifically, analogues were synthesized in which the C2 substituent was the following: (1H-imidazol-1-yl)methyl, (N-morpholinyl)methyl, cyclopentylmethyl, cyclohexylmethyl, or (norborn-2-yl)methyl. Compounds with the cyclic system in the C2 substituent being a carbocyclic ring, especially cyclohexyl or norborn-2-yl, and the N1 substituent being a ring-substituted benzyl group, especially 4-chlorobenzyl or 4-bromobenzyl, best exhibited the target criteria of high potency and selectivity toward inhibition of HO-2. The new candidates should be useful pharmacological tools and may have therapeutic applications.

Solid-state ¹7O NMR spectroscopy of paramagnetic coordination compounds.

High-quality solid-state (17)O (I=5/2) NMR spectra can be successfully obtained for paramagnetic coordination compounds in which oxygen atoms are directly bonded to the paramagnetic metal centers. For complexes containing V(III) (S=1), Cu(II) (S=1/2), and Mn(III) (S=2) metal centers, the (17)O isotropic paramagnetic shifts were found to span a range of more than 10,000 ppm. In several cases, high-resolution (17)O NMR spectra were recorded under very fast magic-angle spinning (MAS) conditions at 21.1 T. Quantum-chemical computations using density functional theory (DFT) qualitatively reproduced the experimental (17)O hyperfine shift tensors.

Acidity and hydrogen exchange dynamics of iron(II)-bound nitroxyl in aqueous solution.

Nitroxyl-iron(II) (HNO-Fe(II)) complexes are often unstable in aqueous solution, thus making them very difficult to study. Consequently, many fundamental chemical properties of Fe(II)-bound HNO have remained unknown. Using a comprehensive multinuclear ((1)H, (15)N, (17)O) NMR approach, the acidity of the Fe(II)-bound HNO in [Fe(CN)5(HNO)](3-) was investigated and its pK(a) value was determined to be greater than 11. Additionally, HNO undergoes rapid hydrogen exchange with water in aqueous solution and this exchange process is catalyzed by both acid and base. The hydrogen exchange dynamics for the Fe(II)-bound HNO have been characterized and the obtained benchmark values, when combined with the literature data on proteins, reveal that the rate of hydrogen exchange for the Fe(II)-bound HNO in the interior of globin proteins is reduced by a factor of 10(6).

Solid-state 17O NMR of pharmaceutical compounds: salicylic acid and aspirin.

We report solid-state NMR characterization of the (17)O quadrupole coupling (QC) and chemical shift (CS) tensors in five site-specifically (17)O-labeled samples of salicylic acid and o-acetylsalicylic acid (Aspirin). High-quality (17)O NMR spectra were obtained for these important pharmaceutical compounds under both static and magic angle spinning (MAS) conditions at two magnetic fields, 14.0 and 21.1 T. A total of 14 (17)O QC and CS tensors were experimentally determined for the seven oxygen sites in salicylic acid and Aspirin. Although both salicylic acid and Aspirin form hydrogen bonded cyclic dimers in the solid state, we found that the potential curves for the concerted double proton transfer in these two compounds are significantly different. In particular, while the double-well potential curve in Aspirin is nearly symmetrical, it is highly asymmetrical in salicylic acid. This difference results in quite different temperature dependencies in (17)O MAS spectra of the two compounds. A careful analysis of variable-temperature (17)O MAS NMR spectra of Aspirin allowed us to obtain the energy asymmetry (ΔE) of the double-well potential, ΔE = 3.0 ± 0.5 kJ/mol. We were also able to determine a lower limit of ΔE for salicylic acid, ΔE > 10 kJ/mol. These asymmetrical features in potential energy curves were confirmed by plane-wave DFT computations, which yielded ΔE = 3.7 and 17.8 kJ/mol for Aspirin and salicylic acid, respectively. To complement the solid-state (17)O NMR data, we also obtained solid-state (1)H and (13)C NMR spectra for salicylic acid and Aspirin. Using experimental NMR parameters obtained for all magnetic nuclei present in salicylic acid and Aspirin, we found that plane-wave DFT computations can produce highly accurate NMR parameters in well-defined crystalline organic compounds.

Variable-temperature 17O NMR studies allow quantitative evaluation of molecular dynamics in organic solids.

We report a comprehensive variable-temperature solid-state (17)O NMR study of three (17)O-labeled crystalline sulfonic acids: 2-aminoethane-1-sulfonic acid (taurine, T), 3-aminopropane-1-sulfonic acid (homotaurine, HT), and 4-aminobutane-1-sulfonic acid (ABSA). In the solid state, all three compounds exist as zwitterionic structures, NH(3)(+)-R-SO(3)(-), in which the SO(3)(-) group is involved in various degrees of O···H-N hydrogen bonding. High-quality (17)O NMR spectra have been obtained for all three compounds under both static and magic angle spinning (MAS) conditions at 21.1 T, allowing the complete set of (17)O NMR tensor parameters to be measured. Assignment of the observed (17)O NMR parameters to the correct oxygen sites in the crystal lattice was achieved with the aid of DFT calculations. By modeling the temperature dependence of (17)O NMR powder line shapes, we have not only confirmed that the SO(3)(-) groups in these compounds undergo a 3-fold rotational jump mechanism but also extracted the corresponding jump rates (10(2)-10(5) s(-1)) and the associated activation energies (E(a)) for this process (E(a) = 48 ± 7, 42 ± 3, and 45 ± 1 kJ mol(-1) for T, HT, and ABSA, respectively). This is the first time that SO(3)(-) rotational dynamics have been directly probed by solid-state (17)O NMR. Using the experimental activation energies for SO(3)(-) rotation, we were able to evaluate quantitatively the total hydrogen bond energy that each SO(3)(-) group is involved in within the crystal lattice. The activation energies also correlate with calculated rotational energy barriers. This work provides a clear illustration of the utility of solid-state (17)O NMR in quantifying dynamic processes occurring in organic solids. Similar studies applied to selectively (17)O-labeled biomolecules would appear to be very feasible.

Multinuclear solid-state nuclear magnetic resonance and density functional theory characterization of interaction tensors in taurine.

A variety of experimental solid-state nuclear magnetic resonance (NMR) techniques has been used to characterize each of the elements in 2-aminoethane sulfonic acid (taurine). A combination of (15)N cross-polarization magic angle spinning (CPMAS), (14)N ultrawideline, and (14)N overtone experiments enabled a determination of the relative orientation of the nitrogen electric field gradient and chemical shift tensors. (17)O spectra recorded from an isotopically enriched taurine sample at multiple magnetic fields allowed the three nonequivalent oxygen sites to be distinguished, and NMR parameters calculated from a neutron diffraction structure using density functional theory allowed the assignment of the (17)O parameters to the correct crystallographic sites. This is the first time that a complete set of (17)O NMR tensors are reported for a sulfonate group. In combination with (1)H and (13)C MAS spectra, as well as a previously reported (33)S NMR study, this provides a very broad set of NMR data for this relatively simple organic molecule, making it a potentially useful structure on which to test DFT calculation methods (particularly for the quadrupolar nuclei (14)N, (17)O, and (33)S) or NMR crystallography approaches.

Investigation of the noncovalent interactions between anti-amyloid agents and amyloid beta peptides by ESI-MS.

This paper describes an efficient and reproducible screening method for identifying low molecular weight compounds that bind to amyloid beta peptides (Abeta) peptides using electrospray ionization mass spectrometry (ESI-MS). Low molecular weight compounds capable of interacting with soluble Abeta may be able to modulate/inhibit the Abeta aggregation process and serve as potential disease-modifying agents for AD. The present approach was used to rank the binding affinity of a library of compounds to Abeta1-40 peptide. The results obtained show that low molecular weight compounds bind similarly to Abeta1-42, Abeta1-40, as well as Abeta1-28 peptides and they underline the critical role of Abeta peptide charge motif in binding at physiological pH. Finally, some elements of structure-activity relationship (SAR) involved in the binding affinity of homotaurine to soluble Abeta peptides are discussed.

Targeting soluble Abeta peptide with Tramiprosate for the treatment of brain amyloidosis.

Amyloid beta-peptide (Abeta) is a major constituent of senile plaques in Alzheimer's disease (AD). Neurotoxicity results from the conformational transition of Abeta from random-coil to beta-sheet and its oligomerization. Among a series of ionic compounds able to interact with soluble Abeta, Tramiprosate (3-amino-1-propanesulfonic acid; 3APS; Alzhemedtrade mark) was found to maintain Abeta in a non-fibrillar form, to decrease Abeta(42)-induced cell death in neuronal cell cultures, and to inhibit amyloid deposition. Tramiprosate crosses the murine blood-brain barrier (BBB) to exert its activity. Treatment of TgCRND8 mice with Tramiprosate resulted in significant reduction (approximately 30%) in the brain amyloid plaque load and a significant decrease in the cerebral levels of soluble and insoluble Abeta(40) and Abeta(42) (approximately 20-30%). A dose-dependent reduction (up to 60%) of plasma Abeta levels was also observed, suggesting that Tramiprosate influences the central pool of Abeta, changing either its efflux or its metabolism in the brain. We propose that Tramiprosate, which targets soluble Abeta, represents a new and promising therapeutic class of drugs for the treatment of AD.