The structure activity relationships of N-(3-acyloxy-2-benzylpropyl)-N′-4-[(methylsulfonylamino)benzyl] thioureas, which represent simplified RTX-based vanilloids, were investigated by varying the distances between the four principal pharmacophores and assessing binding and antagonistic activity on rTRPV1. The analysis indicated that a 3-pivaloyloxy-2-benzylpropyl C-region conferred the best potency in binding affinity and antagonism. The molecular modeling of this best template with the tetrameric homology model of rTRPV1 was performed to identify its binding interactions with the receptor.
Vanilloid Receptor 1; TRPV1; antagonist; resiniferatoxin; molecular modeling; capsaicin
A series of TRPV1 agonists with amide, reverse amide, and thiourea groups in the B-region and their corresponding α-methylated analogues were investigated. Whereas the α–methylation of the amide B-region enhanced the binding affinities and potencies as agonists, that of the reverse amide and thiourea led to a reduction in receptor affinity. The analysis indicated that proper hydrogen bonding as well as steric effects in the B-region are critical for receptor binding.
Vanilloid Receptor 1; TRPV1; agonist; capsaicin
This Letter describes the synthesis and structure–activity relationships (SAR) of isoform-selective PLD inhibitors. By virtue of the installation of a 1,3,8-triazaspiro[4,5]decan-4-one privileged structure, PLD inhibitors with nanomolar potency and an unprecedented 40-fold selectivity for PLD2 over PLD1 were developed. Interestingly, SAR for this diverged from our earlier efforts, and dual PLD1/2 inhibitors were also discovered within this series.
PLD; Phospholipase; Cancer; Privileged structure
Group II metabotropic glutamate receptors (mGluRs) have been implicated in a variety of neurological and psychiatric disorders in recent pathological studies. As a noninvasive medical imaging technique and a powerful tool in neurological research, positron emission tomography (PET) offers the possibility to visualize and study group II mGluRs in vivo under physiologic and pathologic conditions. We synthesized a PET tracer, (S,S,S)-2-(2-carboxycyclopropyl)-2-(3-[11C]methoxyphenethyl) glycine dimethyl ester ([11C]CMGDE), as a prodrug for group II mGluRs, and studied its preliminary biological properties in Sprague-Dawley rats to visualize group II mGluRs. The microPET studies demonstrated that [11C]CMGDE readily penetrated into the brain and upon entering into brain the radiotracer generated from [11C]CMGDE had fast reversible binding in the group II mGluRs rich regions including striatum, hippocampus and different cortical areas. Blocking studies with LY341495 showed 20–30% decrease of binding of the radiotracer generated from [11C]CMGDE in all brain areas with the highest decrease in the striatum 31.5 ± 3.2%. The results show [11C]CMGDE is the first PET tracer that is brain penetrating and can be used to image group II mGluRs in vivo.
group II mGluRs; mGluR2; mGluR3; PET; prodrug
We have synthesized and evaluated a series of 1,4-disubstituted-triazole derivatives for inhibition of the rat NaV1.6 sodium channel isoform, an isoform thought to play an important role in controlling neuronal firing. Starting from a series of 2,4(1H)-diarylimidazoles previously published, we decided to extend the SAR study by replacing the imidazole with a different heterocyclic scaffold and by varying the aryl substituents on the central aromatic ring. The 1,4-disubstituted 1,2,3-triazoles were prepared employing the copper-catalyzed azide-alkyne cycloaddition (CuAAC). Many of the new molecules were able to block the rNav1.6 currents at 10 µM by over 20%, displaying IC50 values ranging in the low micromolar, thus indicating that triazole can efficiently replace the central heterocyclic core. Moreover, the introduction of a long chain at C4 of the central triazole seems beneficial for increased rNav1.6 current block, whereas the length of N1 substituent seems less crucial for inhibition, as long as a phenyl ring is not direcly connected to the triazole. These results provide additional information on the structural features necessary for block of the voltage-gated sodium channels. These new data will be exploited in the preparation of new compounds and could result in potentially useful AEDs.
sodium channels; patch clamp electrophysiology; 1,4-triazoles; rNaV1.6; click chemistry
We report the optimization of a series of non-MPEP site metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs) based on a simple acyclic ether series. Modifications led to a gain of MPEP site interaction through incorporation of a chiral amide in conjunction with a nicotinamide core. A highly potent PAM, 8v (VU0404251), was shown to be efficacious in a rodent model of psychosis. These studies suggest that potent PAMs within topologically similar chemotypes can be developed to preferentially interact or not interact with the MPEP allosteric binding site.
Metabotropic glutamate receptor 5; mGlu5; Positive allosteric modulator (PAM); Non-MPEP
Fluoroquinolone antibiotics have been a mainstay in the treatment of bacterial diseases. The most notable representative, ciprofloxacin, possesses potent antimicrobial activity; however, a rise in resistance to this agent necessitates development of novel derivatives to prolong the clinical lifespan of these antibiotics. Herein we have synthesized and analyzed the antimicrobial properties of a library of N-acylated ciprofloxacin analogues. We find that these compounds are broadly effective against Gram-positive and Gram-negative bacteria, with many proving more effective than the parental drug, and several possessing MICs ≤ 1.0 µg/ml against methicillin-resistant Staphylococcus aureus and Bartonella species. An analysis of spontaneous mutation frequencies reveals very low potential for resistance in MRSA compared to existing fluoroquinolones. Mode of action profiling reveals that modification of the piperazinyl nitrogen by acylation does not alter the effect of these molecules towards their bacterial target. We also present evidence that these N-acylated compounds are highly effective at killing intracellular gbacteria, suggesting the suitability of these antibiotics for therapeutic treatment.
N-Acyl ciprofloxacins; Staphylococcus aureus; MRSA; Antimicrobial activity; Antibiotic resistance
Fragment based drug discovery remains a successful tool for pharmaceutical lead discovery. Although based upon the principle of thermodynamic additivity, the underlying thermodynamic basis is poorly understood. A thermodynamic additivity analysis was performed using stromelysin-1 and a series of biphenyl hydroxamate ligands identified through fragment additivity. Our studies suggest that, in this instance, additivity arises from enthalpic effects, while interaction entropies are unfavorable; this thermodynamic behavior is masked by proton transfer. Evaluation of the changes in constant pressure heat capacities during binding suggest that solvent exclusion from the binding site does not account for the dramatic affinity enhancements observed.
Additivity; Fragment based drug design; Stromelysin-1; Matrix metalloproteinase-3
This Letter describes the synthesis and structure–activity-relationships (SAR) of isoform-selective PLD inhibitors. By virtue of the installation of alternative halogenated piperidinyl benzimidazolone privileged structures, in combination with a key (S)-methyl group, novel PLD inhibitors with low nM potency and unprecedented levels of PLD1 isoform selectivity (~1700-fold) over PLD2 were developed.
Phospholipase D; Cancer; Isoform; PLD1; PLD2
The efficacy of agents that alkylate the O-6 position of guanine is inhibited by O6-alkylguanine-DNA alkyltransferase (AGT) which removes these lesions from the tumor DNA. To increase differential toxicity, inhibitors must selectively deplete AGT in tumors, while sparing normal tissues where this protein serves a protective function. A newly synthesized prodrug of the AGT inhibitor O6-benzylguanine (O6-BG) with an α,α-dimethyl-4-nitrobenzyloxycarbonyl moiety masking the essential 2-amino group has demonstrated the feasibility of targeting hypoxic regions that are unique to solid tumors, for drug delivery. However, these modifications resulted in greatly decreased solubility. Recently, new potent global AGT inhibitors with improved formulatability such as O6-[(3-aminomethyl)benzylguanine (1) have been developed. However, acetylamino (N-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)acetamide) (2) exhibits a pronounced decrease in activity. Thus, 1 would be inactivated by N-acetylation and probably N-glucuronidation. To combat potential conjugational inactivation while retaining favorable solubility, we synthesized 6-((3-((dimethylamino)methyl)benzyl)oxy)-9H-purin-2-amine (3) in which the 3-aminomethyl moiety is protected by methylation; and to impart tumor selectivity we synthesized 2-(4-nitrophenyl)propan-2-yl(6-((3-((dimethylamino)methyl)benzyl)oxy)-9H-purin-2-yl)carbamate (7), a hypoxia targeted prodrug of 3 utilizing an α,α-dimethyl-4-nitrobenzyloxycarbonyl moiety. Consistent with this design, 7 demonstrates both hypoxia selective conversion by EMT6 cells of 7 to 3 and hypoxic sensitization of AGT containing DU145 cells to the cytotoxic actions of laromustine, while exhibiting improved solubility.
Bacterial resistance to β-lactam antibiotics caused by class B metallo-β-lactamases (MBL), especially for certain hospital-acquired, Gram-negative pathogens, poses a significant threat to public health. We report several 2-substituted 4,5-dihydrothiazole-4-carboxylic acids to be novel MBL inhibitors. Structure activity relationship (SAR) and molecular modeling studies were performed and implications for further inhibitor design are discussed.
Metallo-β-lactamase; Inhibitor; Rational design; Molecular modeling
Peroxisome proliferator-activated receptor alpha (PPAR-α) is a ligand-activated nuclear receptor transcription factor that regulates the fatty acid β-oxidation. An in vitro assay identified the p-methoxy phenyl ureido thiobutyric acid derivative KSM-01 (IC50=0.28±0.09 nM) having a higher affinity to activate PPAR-α than the PPAR-α agonist GW7647 ((IC50=0.46±0.19 nM). In this study, we report the synthesis and initial in vivo evaluation of [11C]KSM-01. The radiosynthesis was carried out by first alkylating the corresponding p-phenol precursor with [11C]MeI in DMF using NaOH, followed by deprotection of the t-butyl ester group by TFA, yielding [11C]KSM-01. SUV analysis of dynamic micro PET/CT imaging data showed that [11C]KSM-01 accumulation was ~2.0-fold greater in cardiac-specific PPAR-α overexpressing transgenic mice compared to wild-type littermates. The post-PET biodistribution studies were consistent with these results and demonstrated 2.5-fold greater radiotracer uptake in the heart of transgenic mice compared to the wild-type littermates. These results demonstrate the potential utility of PPAR-α agonists as PET radiopharmaceuticals.
peroxisome proliferator-activated receptor alpha; (PPAR-α); β-oxidation; PET imaging; Cardiomyopathy; Ureido thioisobutyric acid (TiBA)
2-Alkynoic fatty acids display antimycobacterial, antifungal, and pesticidal activities but their antiprotozoal activity has received little attention. In this work we synthesized the 2-octadecynoic acid (2-ODA), 2-hexadecynoic acid (2-HDA), and 2-tetradecynoic acid (2-TDA) and show that 2-ODA is the best inhibitor of the Leishmania donovani DNA topoisomerase IB enzyme (LdTopIB) with an EC50 = 5.3 ± 0.7 μM. The potency of LdTopIB inhibition follows the trend 2-ODA> 2-HDA> 2-TDA, indicating that the effectiveness of inhibition depends on the fatty acid carbon chain length. All of the studied 2-alkynoic fatty acids were less potent inhibitors of the human topoisomerase IB enzyme (hTopIB) as compared to LdTopIB. 2-ODA also displayed in vitro activity against Leishmania donovani (IC50 = 11.0 μM), but it was less effective against other protozoa, Trypanosoma cruzi (IC50 = 48.1 μM) and T. brucei rhodesiense (IC50 = 64.5 μM). The antiprotozoal activity of the 2-alkynoic fatty acids, in general, followed the trend 2-ODA> 2-HDA> 2-TDA. The experimental information gathered so far indicates that 2-ODA is a promising antileishmanial compound.
Acetylenic fatty acids; Leishmania donovani; 2-octadecynoic acid; topoisomerase IB
This Letter describes the synthesis and SAR of two mGluR4 positive allosteric modulator leads, 6 and 7. VU001171 (6) represents the most potent (EC50 = 650 nM), efficacious (141% Glu Max) and largest fold shift (36-fold) of any mGluR4 PAM reported to date. However, this work highlights the challenges in hit-to-lead for mGluR4 PAMs, with multiple confirmed HTS hits displaying little or no tractable SAR.
mGluR4; PAM; Positive allosteric modulator; Parkinson’s Disease
The site-specific incorporation of unnatural amino acids (UAAs) into proteins in bacteria is made possible by the evolution of aminoacyl-tRNA synthetases that selectively recognize and aminoacylate the amino acid of interest. Recently we have discovered that some of the previously evolved aaRSs display a degree of polyspecificity and are capable of recognizing multiple UAAs. Herein we report the polyspecificity of an aaRS evolved to encode a comarin containing amino acid. This polyspecificity was then exploited to introduce several UAAs into the fluorophore of GFP, altering its photophysical properties.
Unnatural amino acids; Polyspecificity; Green Fluorescence Protein; Aminoacyl-tRNA Synthetase; Fluorescence modulation
We investigated the use of infrared vibrational frequency of ligands as a potential novel molecular descriptor in three different molecular target and chemical series. The vibrational energy of a ligand was approximated from the sum of infrared (IR) absorptions of each functional group within a molecule and normalized by its molecular weight (MDIR). Calculations were performed on a set of 4-aminoquinazolines with similar docking scores for the VEGFR2/KDR receptor. 4-Aminoquinazolines with MDIR values ranging 192–196 provided compounds with KDR inhibitory activity. The correlation of KDR inhibitory activity was similarly observed in a separate chemical series, the pyrazolo[1,5-a]pyrimidines. Initial exploration of this molecular descriptor supports a tool for rapid lead optimization in the 4-aminoquinazoline chemical series and a potential method for scaffold hopping in pursuit of new inhibitors.
Attachment of DOTA to a novel monofluoro-cyclooctyne facilitates bioconjugation to an azide-modified peptide via Cu-free click chemistry. The resulting conjugate was radiolabeled with 111In to afford a potential targeted molecular imaging agent with high specific activity and in excellent radiochemical purity.
DOTA; copper-free; click chemistry; peptides; chelators; bioconjugates
Substituted ureas with a carboxylic acid ester as a secondary pharmacophore are potent soluble epoxide hydrolase (sEH) inhibitors. Although the ester substituent imparts better physical properties, such compounds are quickly metabolized to the corresponding less potent acids. Toward producing biologically active ester compounds, a series of esters were prepared and evaluated for potency on the human enzyme, stability in human liver microsomes, and physical properties. Modifications around the ester function enhanced in vitro metabolic stability of the ester inhibitors up to 32-fold without a decrease in inhibition potency. Further, several compounds had improved physical properties.
sEH; sEH inhibitors; substituted urea-ester derivatives; metabolic stability
A number of (1H-1,2,3-triazol-1-yl)benzo[d]thiazoles were synthesized utilizing a versatile Cu-catalyzed azide-alkyne click reaction (CuAAC) on tautomeric benzo[4,5]thiazolo[3,2-d]tetrazole (1) and 2-azidobenzo[d]thiazole (2) starting materials. Moreover, one of the resulting products of this investigation, triazolbenzo[d]thiazole 22, was found to possess significant neuroprotective activity in human neuroblastoma (SH-SY5Y) cells.
Click chemistry; Triazolbenzo[d]thiazoles; Neuroprotective agents
Mcl-1, an anti-apoptotic member of the Bcl-2 protein family, is overexpressed in a broad range of human cancers and plays a critical role in conferring resistance to chemotherapy. In the course of screening a natural product-like library of sesquiterpenoid analogs, we identified substituted hexahydronaphthalenes that showed activity against the Mcl-1/BimBH3 interaction in vitro. Here, we describe the synthesis of a small library of analogs and their biological evaluation. The most potent inhibitor in the series (19) exhibits an IC50 of 8.3 μM by ELISA and disrupts the interaction between endogenously expressed Mcl-1 and Bim in cultured MDA-MB-468 breast cancer cells.
Mcl-1; Bcl-2; Protein-protein interactions; Small molecule inhibitors; Anticancer agents
A high-throughput screen utilizing a depolarization-triggered thallium influx through KCNQ1 channels was developed and used to screen the MLSMR collection of over 300,000 compounds. An iterative medicinal chemistry approach was initiated and from this effort, ML277 was identified as a potent activator of KCNQ1 channels (EC50 = 260 nM). ML277 was shown to be highly selective against other KCNQ channels (>100-fold selectivity versus KCNQ2 and KCNQ4) as well as against the distantly related hERG potassium channel.
KCNQ1 activator; MLPCN probe; Potassium channels; Voltage-gated ion channels; ML277
A series of 3,5-bis (4-hydroxyphenyl) isoxazoles bearing a styryl/alkyl vinyl group at the 4-position were prepared and evaluated as ligands for the estrogen receptor-alpha (ERα). The target compounds were prepared using the Suzuki reaction to couple an iodo-isoxazole intermediate with a series of styryl/alkenyl boronic acids, followed by O-demethylation. The products were evaluated for their estrogen receptor-α ligand binding domain (ERα-LBD) binding affinity using a competitive binding assay. The 4-(4-hydroxystyryl) derivative 4h displays binding properties similar to those of the previously described pyrazole class of ER ligands, indicating that the ERα-LBD tolerates the presence of the added vinyl group at the 4-position of the isoxazole ring.
Estrogen receptors; isoxazoles; synthesis; Suzuki coupling reaction; receptor binding; conformations
Targeting and inhibiting CMG2 (Capillary Morphogenesis Gene protein 2) represents a new strategy for therapeutic agents for cancer and retinal diseases due to CMG2’s role in blood vessel growth (angiogenesis). A high throughput FRET (Förster Resonance Energy Transfer) assay was developed for the identification of CMG2 inhibitors as anti-angiogenetic agents. Bioassay-guided separation led to the isolation and identification of two new compounds (1 and 2) from CR252M, an endophytic fungus Coccomyces proteae collected from a Costa Rican rainforest, and one known compound (3) from CR1207B (Aurapex penicillata). Secondary in vitro assays indicated anti-angiogenic activity. Compound 3 inhibited the endothelial cell migration at 52 µM, but did not show any endothelial cell antiproliferative effect at 156 µM. The structure of the two new compounds, A (1) and B (2), were elucidated on the basis of extensive spectroscopic analysis, including 1D and 2D NMR experiments.
Fungus; Coccomyces proteae; Aurapex penicillata; CMG2; Phenolic
In vivo imaging of voltage-gated sodium channels (VGSCs) can potentially provide insights into the activation of neuronal pathways and aid the diagnosis of a number of neurological diseases. The iminodihydroquinoline WIN17317-3 is one of the most potent sodium channel blockers reported to date and binds with high affinity to VGSCs throughout the rat brain. We have synthesized a 125I-labeled analogue of WIN17317-3 and evaluated the potential of the tracer for imaging of VGSCs with SPECT. Automated patch clamp studies with CHO cells expressing the Nav1.2 isoform and displacement studies with [3H]BTX yielded comparable results for the non-radioactive iodinated iminodihydroquinoline and WIN17317-3. However, the 125I-labeled tracer was rapidly metabolized in vivo, and suffered from low brain uptake and high accumulation of radioactivity in the intestines. The results suggest that iminodihydroquinolines are poorly suited for tracer development.
SPECT; Voltage-gated sodium channel; WIN17317-3; Iodine-125; Imaging
We describe here the identification of non-peptidic vinylsulfones that inhibit parasite cysteine proteases in vitro and inhibit the growth of T. brucei brucei parasites in culture. A high resolution (1.75Å) co-crystal structure of 8a bound to cruzain reveals how the non-peptidic P2/P3 moiety in such analogs bind the S2 and S3 subsites of the protease, effectively recapitulating important binding interactions present in more traditional peptide-based protease inhibitors and natural substrates.