Heparins can be contaminated with oversulfated chondroitin sulfate, OSCS, the impurity being linked to adverse clinical events that certain lots of heparins have had on humans. Here, we propose labeling of the N-acetyl peaks in ¹H NMR spectra of heparins with the parameter D_t, describing the translational diffusion coefficient available from DOSY NMR. We show how DOSY can be applied as a routine method for screening the lots of heparins for obtaining the impurity profile when using ¹H NMR.

A series of tacrine−NO donor hybrid compounds are synthesized and evaluated for cholinesterase inhibitory activity, cognition improving activity, and hepatotoxicity. The pharmacological results indicate that hybrid compounds 1, 2, and 3a potently inhibit cholinesterase in vitro and significantly improve the scopolamine-induced cognition impairment, whereas an analogue (3h) of 2 without the NO donor moiety does not. Compared to tacrine, 1 and 2 show much less hepatotoxicity. Molecular modeling studies suggest that 2 may interact with the catalytic and the peripheral anionic site of acetylcholinesterase.

Inhibitor of secreted frizzled related protein-1 (sFRP-1) would be a novel potential osteogenic agent, since loss of sFRP-1 affects osteoblast proliferation, differentiation, and activity, resulting in improved bone mineral density, quality, and strength. We have identified small molecule diarylsulfone sulfonamide derivatives as sFRP-1 inhibitors. Structure−activity relationship generated for various regions of the scaffold was utilized to improve the biochemical profile, resulting in the identification of potent selective analogues, such as 16 with desirable pharmaceutical profile.

Phosphodiesterase 4 catalyzes the hydrolysis of cyclic AMP and is a target for the development of anti-inflammatory agents. We have designed and synthesized a series of phenyl alkyl ketones as PDE4 inhibitors. Among them, 13 compounds were identified as having submicromolar IC₅₀ values. The most potent compounds have IC₅₀ values of in the mid- to low-nanomolar range. Compound 5v also showed preference for PDE4 with selectivity of >2000-fold over PDE7, PDE9, PDE2, and PDE5. Docking of 5v, 5zf, and 5za into the binding pocket of the PDE4 catalytic domain revealed a similar binding profile to PDE4 with rolipram except that the fluorine atoms of the difluoromethyl groups of 5v, 5za, and 5zf are within a reasonable range for hydrogen bond formation with the amide hydrogen of Thr 333 and the long alkyl chain bears additional van der Waals interactions with His 160, Asp 318, and Tyr 159.

In prior investigation, we discovered that (3′R,4′R)-3-cyanomethyl-4-methyl-3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (4, 3-cyanomethyl-4-methyl-DCK) showed promising anti-HIV activity. In these current studies, we developed and optimized successfully a practical 10-step synthesis for scale-up preparation to increase the overall yield of 4 from 7.8% to 32%. Furthermore, compound 4 exhibited broad-spectrum anti-HIV activity against wild-type and drug-resistant viral infection of CD4+ T cell lines as well as peripheral blood mononuclear cells by both laboratory-adapted and primary HIV-1 isolates with distinct subtypes and tropisms. Compound 4 was further subjected to in vitro and in vivo pharmacokinetic studies. These studies indicated that 4 has moderate cell permeability, moderate oral bioavailability, and low systemic clearance. These results suggest that 4 should be developed as a promising anti-HIV agent for development as a clinical trial candidate.

Cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. Their stability derives from a head-to-tail cyclized backbone coupled with a cystine knot arrangement of three-conserved disulfide bonds. Taking advantage of this stable framework we developed novel VEGF-A antagonists by grafting a peptide epitope involved in VEGF-A antagonism onto the stable cyclotide framework. Antagonists of this kind have potential therapeutic applications in diseases where angiogenesis is an important component of disease progression, including cancer and rheumatoid arthritis. A grafted analogue showed biological activity in an in vitro VEGF-A antagonism assay at low micromolar concentration and the in vitro stability of the target epitope was markedly increased using this approach. In general, the stabilization of bioactive peptide epitopes is a significant problem in medicinal chemistry and in the current study we have provided insight into one approach to stabilize these peptides in a biological environment.

In silico screening is routinely used in the drug discovery process to predict whether each molecule in a database has a function of interest, such as inhibitory activity for a target protein. However, drugs generally have multiple functions including adverse effects. In order to obtain small molecules with desirable physiological effects, it is useful to simultaneously predict as many functions as possible. We employed Support Vector Machine to build classification models for 125 molecular functions, derived from the MDDR database, which showed higher kappa statistics (0.775 on average) than those of predictions by Tanimoto similarity (0.708). By analyzing the patterns of the predicted values (functional profiles) of 871 marketed drugs, we demonstrated its applications to indication discovery, clustering of drugs, and detection of molecular actions related to adverse effects. The results showed that functional profiling can be a useful tool for identifying the multifunctionality or adverse effects of small molecules.

We report herein the synthesis of a series of 19 2-hydroxyisoquinoline-1,3(2H,4H)-dione derivatives variously substituted at position 7 aimed at inhibiting selectively two-metal ion catalytic active sites. The compounds were tested against HIV-1 reverse transcriptase (RT) polymerase, HIV-1 RT ribonuclease H (RNase H), and HIV-1 integrase (IN). Most compounds displayed poor inhibition of RT polymerase even at 50 μM. The majority of the synthesized compounds inhibited RNase H and IN at micromolar concentrations, and some of them were weakly selective for IN. Surprisingly, two new hits were discovered, which displayed a high selectivity for IN with submicromolar IC₅₀ values. These enzymatic inhibitory properties may be related to the metal binding abilities of the compounds. Physicochemical studies were consistent with a 1/1 stoichiometry of the magnesium complexes in solution, and the metal complexation was strictly dependent on the enolization abilities of the compounds. Unfortunately, all tested compounds exhibited high cellular cytotoxicity in cell culture which limits their applications as antiviral agents.

Aminomethylation of 9b,10-dihydro-1H-indolo[1,7:4,5,6]pyrrolo[3,4:2,3][1,4]diazepino-[1,7-a]indole-1,3(2H)-diones or 1H-indolo[1,7:4,5,6]pyrrolo[3,4:2,3][1,4]diazepino[1,7-a]indole-1,3(2H)-diones resulted in dialkylaminomethyl derivatives. Alkylation of the nitrogen atom of maleimide moiety of polyannelated diazepines with 1,3-dibromopropane and subsequent reaction with thiourea or its N-alkyl derivatives gave isothiourea-carrying compounds. The compounds containing isothiourea moiety were active against individual human serine/threonine and tyrosine kinases at low micromolar concentrations. Dialkylaminomethyl derivatives of diazepines sensitized Streptomyces lividans with overexpressed aminoglycoside phosphotransferase type VIII (aphVIII) to kanamycin by inhibiting serine/threonine kinase(s) mediated aphVIII phosphorylation.

Urea-based, low molecular weight ligands of glutamate carboxypeptidase II (GCPII) have demonstrated efficacy in various models of neurological disorders and can serve as imaging agents for prostate cancer. To enhance further development of such compounds, we determined X-ray structures of four complexes between human GCPII and urea-based inhibitors at high resolution. All ligands demonstrate an invariant glutarate moiety within the S1′ pocket of the enzyme. The ureido linkage between P1 and P1′ inhibitor sites interacts with the active-site Zn₁²⁺ ion and the side chains of Tyr552 and His553. Interactions within the S1 pocket are defined primarily by a network of hydrogen bonds between the P1 carboxylate group of the inhibitors and the side chains of Arg534, Arg536, and Asn519. Importantly, we have identified a hydrophobic pocket accessory to the S1 site that can be exploited for structure-based design of novel GCPII inhibitors with increased lipophilicity.

Here, we present the synthesis of five novel indoloquinoxaline derivatives and investigate the DNA binding properties of these monomeric as well as dimeric compounds using absorption, fluorescence, and linear dichroism. Several of the mono- and dicationic derivatives presented have previously demonstrated an excellent antiviral effect that is higher than already acknowledged agents against human cytomegalovirus (CMV), herpes simplex virus type 1 (HSV-1), and varicella-zoster virus (VZV). We find that the DNA binding constants of the monomeric and dimeric derivatives are high (∼10⁶) and very high (∼10⁹), respectively. Results from the spectroscopic measurements show that the planar aromatic indoloquinoxaline moieties upon interaction with DNA intercalate between the nucleobases. Furthermore, we use poly(dA-dT)₂ and calf thymus DNA in a competitive binding experiment to show that all our derivatives have an AT-region preference. The findings are important in the understanding of the antiviral effect of these derivatives and give invaluable information for the future optimization of the DNA binding properties of this kind of drugs.

The design and synthesis of a series of urea-based nonpolycyclic aromatic ligands with alkylaminoanilino side chains as telomeric and genomic G-quadruplex DNA interacting agents are described. Their interactions with quadruplexes have been examined by means of fluorescent resonance energy transfer melting, circular dichroism, and surface plasmon resonance-based assays. These validate the design concept for such urea-based ligands and also show that they have significant selectivity over duplex DNA, as well as for particular G-quadruplexes. The ligand−quadruplex complexes were investigated by computational molecular modeling, providing further information on structure−activity relationships. Preliminary biological studies using short-term cell growth inhibition assays show that some of the ligands have cancer cell selectivity, although they appear to have low potency for intracellular telomeric G-quadruplex structures, suggesting that their cellular targets may be other, possibly oncogene-related quadruplexes.

The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors regulating glucose and lipid metabolism. The search for new PPAR ligands with reduced adverse effects with respect to the marketed antidiabetic agents thiazolidinediones (TZDs) and the dual-agonists glitazars is highly desired. We report the crystal structure and activity of the two enantiomeric forms of a clofibric acid analogue, respectively complexed with the ligand-binding domain (LBD) of PPARγ, and provide an explanation on a molecular basis for their different potency and efficacy against PPARγ. The more potent S-enantiomer is a dual PPARα/PPARγ agonist which presents a partial agonism profile against PPARγ. Docking of the S-enantiomer in the PPARα-LBD has been performed to explain its different subtype pharmacological profile. The hypothesis that partial agonists show differential stabilization of helix 3, when compared to full agonists, is also discussed. Moreover, the structure of the complex with the S-enantiomer reveals a new region of the PPARγ-LBD never sampled before by other ligands.

A series of β-carbolin-2-ones and 3,10-dihydro-2H-azepino[3,4-b]indol-1-ones have been designed, synthesized, and evaluated as RET protein kinase inhibitors on the basis of their structural similarity with the prototype indolin-2-one RPI-1. Some β-carbolin-2-ones (structure 2) showed an ability to inhibit RET enzymatic activity in vitro and proliferation of RETC634R oncogene-transformed NIH3T3 cells comparable to that of the reference compound. The docking analysis of the interaction of these compounds with the crystallographic structure of RET tyrosine kinase domain suggested a new binding interaction scheme different from the one proposed during their design. The rigid structure of the compounds of this series represents a new scaffold with potential advantages in the design of RET protein kinase inhibitors.

The meta-vinylhalide fluoroalkyl ester nortropanes 1−4 were synthesized as ligands of the serotonin transporter (SERT) for use as positron emission tomography (PET) imaging agents. In vitro competition binding assays demonstrated that 1−4 have a high affinity for the SERT (K_i values = 0.3−0.4 nM) and are selective for the SERT over the dopamine and norepinephrine transporters (DAT and NET). MicroPET imaging in anesthetized cynomolgus monkeys with [¹⁸F]1−[¹⁸F]4 demonstrated that all four tracers behave similarly with peak uptake in the SERT-rich brain regions achieved after 45−55 min, followed by a steady washout. An awake monkey study was performed with [¹⁸F]1, which demonstrated that the uptake of [¹⁸F]1 was not influenced by anesthesia. Chase studies with the SERT ligand 15 displaced [¹⁸F]1−[¹⁸F]4, but chase studies with the DAT ligand 16 did not displace [¹⁸F]1−[¹⁸F]4 thus indicating that the tracers were binding specifically to the SERT.

N-(4-Hydroxyphenyl)-(5Z,8Z,11Z,14Z)-icosatetra-5,8,11,14-enamide (AM404) is a metabolite of the well-known analgesic paracetamol. AM404 inhibits endocannabinoid cellular uptake, binds weakly to CB₁ and CB₂ cannabinoid receptors, and is formed by fatty acid amide hydrolase (FAAH) in vivo. We prepared three derivatives of this new (endo)cannabinoid using bioisosteric replacement (1), homology (2), and derivatization (3) of the 4-aminophenol moiety in AM404 and tested them against CB₁, CB₂, and FAAH. We found affinities toward both cannabinoid receptors equal to or greater than that of AM404. Shortening the acyl chain from C₂₀ to C₂ led to three new paracetamol analogues: N-(1H-indazol-5-yl)acetamide (5), N-(4-hydroxybenzyl)acetamide (6), and N-(4-hydroxy-3-methoxyphenyl)acetamide (7). Again, 5, 6, and 7 were tested against CB₁, CB₂, and FAAH without significant activity. However, 5 and 7 behaved like inhibitors of cyclooxygenases in whole blood assays. Finally, 5 (50 mg/kg) and 6 (275 mg/kg) displayed analgesic activities comparable to paracetamol (200 mg/kg) in the mouse formalin test.

A series of structurally constrained analogues based on hybrid compounds containing octahydrobenzo[g or f]quinoline moieties were designed, synthesized, and characterized for their binding to dopamine D2 and D3 receptors expressed in HEK-293 cells. Among the newly developed constrained molecules, trans-octahydrobenzo[f]quinolin-7-ol (8) exhibited the highest affinity for D2 and D3 receptors, the (−)-isomer being the eutomer. Interestingly, this hybrid constrained version 8 showed significant affinity over the corresponding nonhybrid version 1 (representing a constrained version of the aminotetralin structure only) when assayed under same conditions (K_i of 49.1 and 14.9 nM for 8 vs 380 and 96.0 nM for 1 at D2 and D3, respectively). Similar results were found with other lead hybrid compounds, indicating a contribution of the piperazine moiety in the observed enhanced affinity. On the basis of the data of new lead constrained derivatives and other lead hybrid derivatives developed by us, a unique pharmacophore model was proposed consisting of three pharmacophoric centers, two with aromatic/hydrophobic and one with cationic features.

Synthesis and identification of novel phenylalkyl isoselenocyanates (ISCs), isosteric selenium analogues of naturally occurring phenylalkyl isothiocyanates (ITCs), as effective cytotoxic and antitumor agents are described. The structure−activity relationship comparison of ISCs with ITCs and effect of the increasing alkyl chain length in inhibiting cancer cell growth were evaluated on melanoma, prostate, breast, glioblastoma, sarcoma, and colon cancer cell lines. IC₅₀ values for ISC compounds were generally lower than their corresponding ITC analogues. Similarly, in UACC 903 human melanoma cells, the inhibition of cell proliferation and induction of apoptosis were more pronounced with ISCs compared to ITCs. Further, ISCs and ITCs effectively inhibited melanoma tumor growth in mice following intraperitoneal xenograft. A similar reduction in tumor size was observed at 3 times lower doses of ISCs compared to corresponding ITCs.

We screened 26 bisphosphonates against a farnesyl diphosphate synthase from Plasmodium vivax, finding a poor correlation between enzyme and cell growth inhibition (R² = 0.06). To better predict cell activity data, we then used a combinatorial descriptor search in which pIC₅₀(cell) = a pIC₅₀(enzyme) + bB + cC + d, where B and C are descriptors (such as SlogP), and a−d are coefficients. R² increased from 0.01 to 0.74 (for a leave-two-out test set of 26 predictions). The method was then further validated using data for nine other systems, including bacterial, viral, and mammalian cell systems. On average, experimental/predicted cell pIC₅₀ correlations increased from R² = 0.28 (for an enzyme-only test set) to 0.70 (for enzyme plus two descriptor test set predictions), while predictions based on scrambled cell activity had no predictive value (R² = 0.13). These results are of interest since they represent a general way to predict cell from enzyme inhibition data, with in three cases, R² values increasing from ∼0.02 to 0.72.

Derangement of cellular immunity is central in the pathophysiology of multiple sclerosis (MS) and is often manifested by abnormal cytokine production. We investigated cytokine secretion in peripheral blood mononuclear cells (PBMC) of 18 MS patients and 15 controls and correlated cytokine polarization with the nature of antigenic stimulus. We synthesized two novel citrullinated peptides, linear [Cit⁹¹, Ala⁹⁶, Cit⁹⁷]MBP₈₇₋₉₉ and cyclo(87−99)[Cit⁹¹, Ala⁹⁶, Cit⁹⁷]MBP₈₇₋₉₉ that resulted from citrullination of 91,97 Arg residues in antagonists, linear [Arg⁹¹, Ala⁹⁶]MBP₈₇₋₉₉ and cyclo(87−99)[Arg⁹¹, Ala⁹⁶]MBP₈₇₋₉₉ peptides. PBMC from MS patients and controls were cultured with citrullinated peptides, and both peptides caused a Th1 polarization in all MS patients studied. In contrast, culture with noncitrullinated MBP peptides resulted in heterogeneous cytokine secretion that differed between individual patients. Thus, citrullination of self-antigens may potentially trigger disease in susceptible individuals. This finding may open new avenues in drug design of new substances that inhibit citrullination and arrest epitope spreading and worsening of MS.

On the basis of the structures of small-molecule hits targeting the HIV-1 gp41, N-(4-carboxy-3-hydroxy)phenyl-2,5-dimethylpyrrole (2, NB-2), and N-(3-carboxy-4-chloro)phenylpyrrole (A₁, NB-64), 42 N-carboxyphenylpyrrole derivatives in two categories (A and B series) were designed and synthesized. We found that 11 compounds exhibited promising anti-HIV-1 activity at micromolar level and their antiviral activity was correlated with their inhibitory activity on gp41 six-helix bundle formation, suggesting that these compounds block HIV fusion and entry by disrupting gp41 core formation. The structure−activity relationship and molecular docking analysis revealed that the carboxyl group could interact with either Arg579 or Lys574 to form salt bridges and two methyl groups on the pyrrole ring were favorable for interaction with the residues in gp41 pocket. The most active compound, N-(3-carboxy-4-hydroxy)phenyl-2,5-dimethylpyrrole (A₁₂), partially occupied the deep hydrophobic pocket, suggesting that enlarging the molecular size of A₁₂ could improve its binding affinity and anti-HIV-1 activity for further development as a small-molecule HIV fusion and entry inhibitor.

From a series of small fragments that was designed to probe the histamine H₄ receptor (H₄R), we previously described quinoxaline-containing fragments that were grown into high affinity H₄R ligands in a process that was guided by pharmacophore modeling. With a scaffold hopping exercise and using the same in silico models, we now report the identification and optimization of a series of quinazoline-containing H₄R compounds. This approach led to the discovery of 6-chloro-N-(furan-3-ylmethyl)2-(4-methylpiperazin-1-yl)quinazolin-4-amine (VUF10499, 54) and 6-chloro-2-(4-methylpiperazin-1-yl)-N-(thiophen-2-ylmethyl)quinazolin-4-amine (VUF10497, 55) as potent human H₄R inverse agonists (pK_i = 8.12 and 7.57, respectively). Interestingly, both compounds also possess considerable affinity for the human histamine H₁ receptor (H₁R) and therefore represent a novel class of dual action H₁R/H₄R ligands, a profile that potentially leads to added therapeutic benefit. Compounds from this novel series of quinazolines are antagonists at the rat H₄R and were found to possess anti-inflammatory properties in vivo in the rat.

In the isomeric series of 12 racemic topologically rigid N-methyl analogues of oxide-bridged phenylmorphans, all but two of the racemates, the ortho- and para-b-oxide-bridged phenylmorphans 20 and 12, have remained to be synthesized. The b-isomers were very difficult to synthesize because of the highly strained 5,6-trans-fused ring junction that had to be formed. Our successful strategy required functionalization of the position para (or ortho) to a fluorine atom on the aromatic ring using an electron-withdrawing nitro group to activate that fluorine. The racemic N-phenethyl analogues 24 and 16 were moderately potent κ-receptor antagonists in the [³⁵S]GTPγS assay. We synthesized the N-phenethyl-substituted oxide-bridged phenylmorphans in the ortho- and para-d-oxide-bridged phenylmorphan series (51 and 52) which had not been previously evaluated using contemporary receptor binding assays to see whether they also have higher affinity for opioid receptors than their N-methyl relatives 46 and 47.

Multitarget drugs have been to be found effective in controlling complex diseases. However, how to design multitarget drugs presents a great challenge. We have developed a computer-assisted strategy to screen for multitarget inhibitors using a combination of molecular docking and common pharmacophore matching. This strategy was successfully applied to screen for dual-target inhibitors against both the human leukotriene A₄ hydrolase (LTA4H-h) and the human nonpancreatic secretory phospholipase A2 (hnps-PLA2). Three compounds screened from the chemical database MDL Available Chemical Directory were found to inhibit these two enzymes at the 10 μM level. Moreover, one synthetic compound matching the common pharmacophores inhibits LTA4H-h and hnps-PLA2 with IC₅₀ values of 35 nM and 7.3 μM, respectively. The common pharmacophore model can also be used to search small molecule databases or collections of existing inhibitors, as well as to guide combinatorial library design to search for ideal multitarget inhibitors.

Calcitonin gene-related peptide (CGRP) is a 37-residue neuropeptide that can be converted to a CGRP₁ receptor antagonist by the truncation of its first seven residues. CGRP(8−37), 1, has a CGRP₁ receptor K_i = 3.2 nM but is rapidly degraded in human plasma (t_1/2 = 20 min). As part of an effort to identify a prolonged in vivo circulating CGRP peptide antagonist, we found that the substitution of multiple residues in the CGRP peptide increased CGRP₁ receptor affinity >50-fold. Ac-Trp-[Arg²⁴,Lys²⁵,Asp³¹,Pro³⁴,Phe³⁵]CGRP(8−37)-NH₂, 5 (K_i = 0.06 nM) had the highest CGRP₁ receptor affinity. Using complimentary in vitro and in vivo metabolic studies, we iteratively identified degradation sites and prepared high affinity analogues with significantly improved plasma stability. Ac-Trp-[Cit^11,18,hArg²⁴,Lys²⁵,2-Nal^27,37,Asp³¹,Oic^29,34,Phe³⁵]CGRP(8−37)-NH₂, 32 (K_i = 3.3 nM), had significantly increased (>100-fold) stability over 1 or 5, with a cynomolgus monkey and human in vitro plasma half-life of 38 and 68 h, respectively.

More than 500 compounds chosen to represent kinase inhibitor space have been screened against a panel of over 200 protein kinases. Significant results include the identification of hits against new kinases including PIM1 and MPSK1, and the expansion of the inhibition profiles of several literature compounds. A detailed analysis of the data through the use of affinity fingerprints has produced findings with implications for biological target selection, the choice of tool compounds for target validation, and lead discovery and optimization. In a detailed examination of the tyrosine kinases, interesting relationships have been found between targets and compounds. Taken together, these results show how broad cross-profiling can provide important insights to assist kinase drug discovery.

The interaction of the chemokine receptor CXCR4 with its ligand CXCL12 is involved in many biological processes such as hematopoesis, migration of immune cells, as well as in cancer metastasis. CXCR4 also mediates the infection of T-cells with X4-tropic HIV functioning as a coreceptor for the viral envelope protein gp120. Here, we describe highly potent, selective CXCR4 inhibitors that block CXCR4/CXCL12 interactions in vitro and in vivo as well as the infection of target cells by X4-tropic HIV.

Type-II kinase inhibitors represent a class of chemicals that trap their target kinases in an inactive, so-called DFG-out state, occupying a hydrophobic pocket adjacent to the ATP binding site. These compounds are often more specific than those that target active DFG-in kinase conformations. Unfortunately, the discovery of novel type-II scaffolds presents a considerable challenge, partially because the lack of compatible kinase structures makes structure-based methods inapplicable. We present a computational protocol for converting multiple available DFG-in structures of various kinases (∼70% of mammalian structural kinome) into accurate and specific models of their type-II bound state. The models, described as deletion-of-loop Asp-Phe-Gly-in (DOLPHIN) kinase models, demonstrate exceptional performance in various inhibitor discovery applications, including compound pose prediction, screening, and in silico activity profiling. Given the abundance of the DFG-in structures, the presented approach opens possibilities for kinome-wide discovery of specific molecules targeting inactive kinase states.

To extend our development of new imaging agents targeting the prostate-specific membrane antigen (PSMA), we have used the versatile intermediate 2-[3-(5-amino-1-carboxy-pentyl)-ureido]-pentanedioic acid (Lys-C(O)-Glu), which allows ready incorporation of radiohalogens for single photon emission computed tomography (SPECT) and positron emission tomography (PET). We prepared 2-[3-[1-carboxy-5-(4-[¹²⁵I]iodo-benzoylamino)-pentyl]-ureido]-pentanedioic acid ([¹²⁵I]3), 2-[3-[1-carboxy-5-(4-[¹⁸F]fluoro-benzoylamino)-pentyl]-ureido]-pentanedioic acid ([¹⁸F]6), and 2-(3-[1-carboxy-5-[(5-[¹²⁵I]iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-pentanedioic acid ([¹²⁵I]8) in 65−80% (nondecay-corrected), 30−35% (decay corrected), and 59−75% (nondecay-corrected) radiochemical yields. Compound [¹²⁵I]3 demonstrated 8.8 ± 4.7% injected dose per gram (%ID/g) within PSMA⁺ PC-3 PIP tumor at 30 min postinjection, which persisted, with clear delineation of the tumor by SPECT. Similar tumor uptake values at early time points were demonstrated for [¹⁸F]6 (using PET) and [¹²⁵I]8. Because of the many radiohalogenated moieties that can be attached via the ε amino group, the intermediate Lys-C(O)-Glu is an attractive template upon which to develop new imaging agents for prostate cancer.

A number of bis(diazeniumdiolates) that we designed to release up to 4 mol of nitric oxide (NO) and that are structural analogues of the NO prodrug and anticancer lead compound O²-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino)diazen-1-ium-1,2- diolate (PABA/NO) were synthesized and studied. A majority of these compounds yielded higher levels of NO, were better inhibitors of proliferation of a number of cancer cell lines, and more rapidly induced substantially increased levels of S-glutathionylation of cellular proteins in comparison with PABA/NO. In most cases, the antiproliferative activity and extents of S-glutathionylation correlated well with levels of intracellular NO release. We report bis(diazeniumdiolates) to be a class of S-glutathionylating agents with potent antiproliferative and S-glutathionylating activity.

A series of compounds containing the 2-amino-1,3-thiazol-4(5H)-one core were found to be potent inhibitors of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). One of our lead compounds from this series activated the human nuclear xenobiotic receptor, pregnane X receptor (PXR). To try and mitigate the PXR activity, we prepared analogues of our lead series that contained polar groups on the right-hand side of the thiazolone. Several analogues containing amides or alcohols appended to the C-5 position of the thiazolone showed a significant reduction in PXR activity. Through these structure−activity efforts, a compound containing a tert-alcohol group off the C-5 position, analogue (S)-33a, was found to have an 11β-HSD1 K_i = 35 nM and negligible PXR activity. Compound (S)-33a was advanced into a pharmacodynamic model in cynomolgus monkeys, where it inhibited adipose 11β-HSD1 activity after being orally administered.

Matrix metalloproteinases (MMPs) and carbonic anhydrases (CAs) are two classes of zinc enzymes with different roles and catalytic targets, such as the degradation of most of the extracellular matrix (ECM) proteins and the regulation of the CO₂/HCO₃⁻ equilibrium in the cells, respectively. Both families have isoforms which were proved to be involved in several stages of carcinogenic processes, and so the selective inhibition of these enzymes might be of interest in cancer therapy. We report herein the design, synthesis, and in vitro evaluation of a series of compounds possessing the iminodiacetic acid as the main backbone and two functional groups attached, namely, the hydroxamic acid and the arylsulfonamide (ArSO₂NH₂) moieties, to enable the inhibition of MMPs and CAs, respectively. These compounds were demonstrated to strongly inhibit both MMPs and CAs, some of them from the nanomolar to subnanomolar range. Furthermore, a docking study for MMPs was reported for the most promising compound in order to investigate its binding interactions with the different MMPs.

This report describes the synthesis of two new cyclic RGD (Arg-Gly-Asp) dimers, 3 (E[G₃-c(RGDfK)]₂) and 4 (G₃-E[G₃-c(RGDfK)]₂), and their corresponding conjugates 5 (HYNIC-E[G₃-c(RGDfK)]₂: HYNIC = 6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl) and 6 (HYNIC-G₃-E[G₃-c(RGDfK)]₂). Integrin α_vβ₃ binding affinities of 5 and 6 were determined by displacement of ¹²⁵I-echistatin bound to U87MG glioma cells. ^99mTc complexes 7 ([^99mTc(5)(tricine)(TPPTS)]: TPPTS = trisodium triphenylphosphine-3,3′,3′′-trisulfonate) and 8 ([^99mTc(6)(tricine)(TPPTS)]) were prepared in high yield and high specific activity. Biodistribution and imaging studies were performed in athymic nude mice bearing U87MG glioma and MDA-MB-435 breast cancer xenografts. It was found that G₃ linkers are particularly useful for increasing integrin α_vβ₃ binding affinity of cyclic RGD dimers and improving the tumor uptake and clearance kinetic of their ^99mTc radiotracers. Complex 8 is a very promising radiotracer for the early detection of integrin α_vβ₃-positive tumors and may have the potential for noninvasive monitoring of tumor growth or shrinkage during antiangiogenic treatment.

Aspirin prodrugs formed by derivatization at the benzoic acid group are very difficult to obtain because the promoiety accelerates the rate of hydrolysis by plasma esterases at the neighboring acetyl group, generating salicylic acid derivatives. By tracing the hydrolysis pattern of the aspirin prodrug isosorbide-2,5-diaspirinate (ISDA) in human plasma solution, we were able to identify a metabolite, isosorbide-2-aspirinate-5-salicylate, that undergoes almost complete conversion to aspirin by human plasma butyrylcholinesterase, making it the most successful aspirin prodrug discovered to date.

CYP2C9 is a significant P450 protein responsible for drug metabolism. With the increased use of heterocyclic compounds in drug design, a rapid and efficient predrug screening of these potential type II binding compounds is essential to avoid adverse drug reactions. To understand binding modes, we use quinoline-4-carboxamide analogues to study the factors that determine the structure−activity relationships. The results of this study suggest that the more accessible pyridine with the nitrogen para to the linkage can coordinate directly with the ferric heme iron, but this is not seen for the meta or ortho isomers. The π−cation interaction of the naphthalene moiety and Arg 108 residue may also assist in stabilizing substrate binding within the active-site cavity. The type II substrate binding affinity is determined by the combination of steric, electrostatic, and hydrophobicity factors; meanwhile, it is enhanced by the strength of lone pair electrons coordination with the heme iron.

Here, we examine the significance that stereochemistry plays within the clinically relevant Janus kinase 3 (Jak3) inhibitor 1 (CP-690,550). A synthesis of all four enantiopure stereoisomers of the drug was carried out and an examination of each compound revealed that only the enantiopure 3R,4R isomer was capable of blocking Stat5 phosphorylation (Jak3 dependent). Each compound was profiled across a panel of over 350 kinases, which revealed a high level of selectivity for the Jak family kinases for these related compounds. Each stereoisomer retained a degree of binding to Jak3 and Jak2 and the 3R,4S and 3S,4R stereoisomers were further revealed to have binding affinity for selected members of the STE7 and STE20 subfamily of kinases. Finally, an appraisal of the minimum energy conformation of each stereoisomer and molecular docking at Jak3 was performed in an effort to better understand each compounds selectivity and potency profiles.

Mechanistic and structural studies have been carried out to investigate the molecular basis for the irreversible inhibition of human MAO-B by mofegiline. Competitive inhibition with substrate shows an apparent K_i of 28 nM. Irreversible inhibition of MAO-B occurs with a 1:1 molar stoichiometry with no observable catalytic turnover. The absorption spectral properties of mofegiline inhibited MAO-B show features (λ_max ≃ 450 nm) unlike those of traditional flavin N(5) or C(4a) adducts. Visible and near-UV circular dichroism spectra of the mofegiline−MAO-B adduct shows a negative peak at 340 nm with an intensity similar to that of N(5) flavocyanine adducts. The X-ray crystal structure of the mofegiline−MAO-B adduct shows a covalent bond between the flavin cofactor N(5) with the distal allylamine carbon atom as well as the absence of the fluorine atom. A mechanism to explain these structural and spectral data is proposed.

The development of selective inhibitors for individual PLA₂ enzymes is necessary in order to target PLA₂-specific signaling pathways, but it is challenging due to the observed promiscuity of known PLA₂ inhibitors. In the current work, we present the development and application of a variety of synthetic routes to produce pentafluoro, tetrafluoro, and trifluoro derivatives of activated carbonyl groups in order to screen for selective inhibitors and characterize the chemical properties that can lead to selective inhibition. Our results demonstrate that the pentafluoroethyl ketone functionality favors selective inhibition of the GVIA iPLA₂, a very important enzyme for which specific, potent, reversible inhibitors are needed. We find that 1,1,1,2,2-pentafluoro-7-phenyl-heptan-3-one (FKGK11) is a selective inhibitor of GVIA iPLA₂ (X_I(50) = 0.0073). Furthermore, we conclude that the introduction of an additional fluorine atom at the α′ position of a trifluoromethyl ketone constitutes an important strategy for the development of new potent GVIA iPLA₂ inhibitors.

Galanin is an endogenous neuropeptide that modulates seizures in the brain. Because this neuropeptide does not penetrate the blood−brain barrier, we designed truncated galanin analogues in which nonessential amino acid residues were replaced by cationic and/or lipoamino acid residues. The analogues prevented seizures in the 6 Hz mouse model of epilepsy following intraperitoneal administration. The most active analogue, Gal-B2 (NAX 5055), contained the -Lys-Lys-Lys(palmitoyl)-Lys-NH₂ motif and exhibited high affinity for galanin receptors (K_i = 3.5 nM and 51.5 nM for GalR1 and GalR2, respectively), logD = 1.24, minimal helical conformation and improved metabolic stability. Structure−activity-relationship analysis suggested that cationization combined with position-specific lipidization was critical for improving the systemic activity of the analogues. Because the anticonvulsant activity of galanin is mediated by the receptors located in hippocampus and other limbic brain structures, our data suggest that these analogues penetrate into the brain. Gal-B2 may lead to development of first-in-class antiepileptic drugs.

Previous studies showed that the mixed monoamine transporter inhibitor (6, RTI-112) reduced cocaine self-administration at a high level of serotonin transporter (5-HTT) occupancy with no detectable dopamine transporter (DAT) occupancy. In this study, a series of 3β-(substituted phenyl)tropane-2β-carboxylic acid methyl esters (7a−g), 3β-(4-methoxyphenyl)tropane-2β-carboxylic acid esters (8a−j), and 3β-(4-methoxyphenyl)-2β-[3-(4′-methylphenyl)isoxazol-5-yl]tropane (9) were synthesized and evaluated for their monoamine transporter binding affinities to identify potent and selective compounds for both the DAT and 5-HTT relative to the norepinephrine transporter (NET). A number of compounds showed high binding affinities for both the DAT and 5-HTT and low affinity for the NET. 3β-(4-Methoxyphenyl)tropane-2β-carboxylic acid 2-(3-iodo-4-aminophenyl)ethyl ester (8i) with an IC₅₀ value of 2.5 nM for the DAT and K_i values of 3.5 and 2040 nM for the 5-HTT and NET, respectively, is the most potent and selective compound for the DAT and 5-HTT relative to the NET in this study.

Imaging of programmed cell death (apoptosis) is important in the assessment of therapeutic response in oncology and for diagnosis in cardiac and neurodegenerative disorders. The executioner caspases 3 and 7 ultimately effect cellular death, thus providing selective molecular targets for in vivo quantification of apoptosis. To realize this potential, we aimed to develop ¹⁸F-labeled isatin sulfonamides with high metabolic stability and moderate lipophilicity while retaining selectivity and affinity for caspase 3/7. A small library of isatins modified with fluorinated aromatic groups and heterocycles was synthesized. A lead compound incorporating 2′-fluoroethyl-1,2,3-triazole was identified with subnanomolar affinity for caspase 3. “Click labeling” provided the ¹⁸F-labeled tracer in 65 ± 6% decay-corrected radiochemical yield from 2-[¹⁸F]fluoroethylazide. The compound showed high stability in vivo with rapid uptake and elimination in healthy tissues and tumor. The novel ¹⁸F-labeled isatin is a candidate radiotracer for further preclinical evaluation for imaging of apoptosis.

Dual inhibition of the prostaglandin (PG) and leukotriene (LT) biosynthetic pathway is supposed to be superior over single interference, both in terms of efficacy and side effects. Here, we present a novel class of dual microsomal PGE₂ synthase-1/5-lipoxygenase (5-LO) inhibitors based on the structure of pirinixic acid [PA, 2-(4-chloro-6-(2,3-dimethylphenylamino)pyrimidin-2-ylthio)acetic acid, compound 1]. Target-oriented structural modification of 1, particularly α substitution with extended n-alkyl or bulky aryl substituents and concomitant replacement of the 2,3-dimethylaniline by a biphenyl-4-yl-methane-amino residue, resulted in potent suppression of mPGES-1 and 5-LO activity, exemplified by 2-(4-(biphenyl-4-ylmethylamino)-6-chloropyrimidin-2-ylthio)octanoic acid (7b, IC₅₀ = 1.3 and 1 μM, respectively). Select compounds also potently reduced PGE₂ and 5-LO product formation in intact cells. Importantly, inhibition of cyclooxygenases-1/2 was significantly less pronounced. Taken together, these pirinixic acid derivatives constitute a novel class of dual mPGES-1/5-LO inhibitors with a promising pharmacologial profile and a potential for therapeutic use.

Pyridine substituted naphthalenes (e.g., I−III) constitute a class of potent inhibitors of aldosterone synthase (CYP11B2). To overcome the unwanted inhibition of the hepatic enzyme CYP1A2, we aimed at reducing the number of aromatic carbons of these molecules because aromaticity has previously been identified to correlate positively with CYP1A2 inhibition. As hypothesized, inhibitors with a tetrahydronaphthalene type molecular scaffold (1−11) exhibit a decreased CYP1A2 inhibition. However, tetralone 9 turned out to be cytotoxic to the human cell line U-937 at higher concentrations. Consequent structural optimization culminated in the discovery of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones (12−26), with 12, a bioisostere of 9, being nontoxic up to 200 μM. The investigated molecules are highly selective toward both CYP1A2 and a wide range of other cytochrome P450 enzymes and show a good pharmacokinetic profile in vivo (e.g., 12 with a peroral bioavailability of 71%). Furthermore, isoquinoline derivative 21 proved to significantly reduce plasma aldosterone levels of ACTH stimulated rats.

γ-Hydroxybutyrate (GHB) is a metabolite of γ-aminobutyric acid (GABA) and has been proposed to function as a neurotransmitter or neuromodulator. GHB is used in the treatment of narcolepsy and is a drug of abuse. GHB binds to both GABA_B receptors and specific high-affinity GHB sites in brain, of which the latter have not been linked unequivocally to function, but are speculated to be GHB receptors. In this study, a series of biaromatic 4-substituted GHB analogues, including 4′-phenethylphenyl, 4′-styrylphenyl, and 4′-benzyloxyphenyl GHB analogues, were synthesized and characterized pharmacologically in a [³H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid ([³H]NCS-382) binding assay and in GABA_A and GABA_B receptor binding assays. The compounds were selective for the high-affinity GHB binding sites and several displayed K_i values below 100 nM. The affinity of the 4-[4′-(2-iodobenzyloxy)phenyl] GHB analogue 17b was shown to reside predominantly with the R-enantiomer (K_i = 22 nM), which has higher affinity than previously reported GHB ligands.

Type 2 diabetes mellitus has reached epidemic proportions; therefore, the search for novel antihyperglycemic drugs is intense. We have discovered that d-xylose increases the rate of glucose transport in a non-insulin-dependent manner in rat and human myotubes in vitro. Due to the unfavorable pharmacokinetic properties of d-xylose we aimed at synthesizing active derivatives with improved parameters. Quantitative structure−activity relationship analysis identified critical hydroxyl groups in d-xylose. These data were used to synthesize various hydrophobic derivatives of d-xylose of which compound 19 the was most potent compound in stimulating the rate of hexose transport by increasing the abundance of glucose transporter-4 in the plasma membrane of myotubes. This effect resulted from the activation of AMP-activated protein kinase without recruiting the insulin transduction mechanism. These results show that lipophilic d-xylose derivatives may serve as prototype molecules for the development of novel antihyperglycemic drugs for the treatment of diabetes.

Aromatic 3,3′-diesters of thiodigalactoside were synthesized in a rapid three-step sequence from commercially available thiodigalactoside and evaluated as inhibitors of cancer- and immunity-related galectins. For each of galectins-1, -3, -7, and -9N-terminal domain, aromatic 3,3′-diesters of thiodigalactoside were found to have affinities in the low micromolar range, which represents a 7−70 fold enhancement over thiodigalactoside itself. No significant improvement was found for galectin-8 N-terminal domain. Two of the compounds were selected for testing in cell culture and were shown to have potent antimigratory effects on human PC-3 prostate and human A549 nonsmall-cell lung cancer cells.

In our attempt to further develop the cycloSal pronucleotide concept, we report on 5-(1-acetoxyvinyl)-cycloSal-d4TMPs as a new type of enzyme-activated pronucleotides. These compounds were converted into 5-acetyl-cycloSal-d4TMPs by (carboxy)esterase cleavage inside the cells. The enzymatic reaction led to the formation of a strong electron-withdrawing substituent that strongly accelerates the chemical hydrolysis of the cycloSal nucleotide to give d4TMP. For some cycloSal-d4TMPs a separation into the diastereomers was achieved. The absolute configuration was assigned by correlation of circular dichroism spectra with similar compounds. Most of the compounds showed complete retention of antiviral activity in TK-deficient CEM/TK⁻ cells, which proves the TK-bypass potential of this approach. Interestingly, (S_P)-isomers of cycloSal phosphate triesters showed better antiviral activity in HIV-2-infected thymidine-kinase deficient CEM/TK⁻ cells than their (R_P)-counterparts.

Vasopressin (AVP) is a hormone that stimulates an increase in water permeability through activation of V2 receptors in the kidney. The analogue of AVP, desmopressin, has proven an effective drug for diseases where a reduction of urine output is desired. However, its peptidic nature limits its bioavailability. We report herein the discovery of potent, nonpeptidic, benzylurea derived agonists of the vasopressin V2 receptor. We describe substitutions on the benzyl group to give improvements in potency and subsequent modifications to the urea end group to provide improvements in solubility and increased oral efficacy in a rat model of diuresis. The lead compound 20e (VA106483) is reported for the first time and has been selected for clinical development.

Per-butanoylated N-acetyl-d-mannosamine (Bu₄ManNAc), a SCFA-hexosamine cancer drug candidate with activity manifest through intact n-butyrate-carbohydrate linkages, reduced the invasion of metastatic MDA-MB-231 breast cancer cells unlike per-butanoylated-d-mannose (Bu₅Man), a clinically tested compound that did not alter cell mobility. To gain molecular-level insight, therapeutic targets implicated in metastasis were investigated. The active compound Bu₄ManNAc reduced both MUC1 expression and MMP-9 activity (via down-regulation of CXCR4 transcription), whereas “inactive” Bu₅Man had counterbalancing effects on these oncogenes. This divergent impact on transcription was linked to interplay between HDACi activity (held by both Bu₄ManNAc and Bu₅Man) and NF-κB activity, which was selectively down-regulated by Bu₄ManNAc. Overall, these results establish a new therapeutic end point (control of invasion) for SCFA-hexosamine hybrid molecules, define relative contributions of molecular players involved in cell mobility and demonstrate that Bu₄ManNAc breaks the confounding link between beneficial HDACi activity and the simultaneous deleterious activation of NF-κB often found in epigenetic drug candidates.

Two series of quinolinquinone derivatives, 2′H-spiro[imidazolidine-4,3′-thieno[2,3-g]quinoline]-2,4′,5,9′-tetraones (2a−n) and 2H-spiro[thieno[2,3-g]quinoline-3,5′-[1,2,4]triazinane]-3′,4,6′,9-tetraones (3a−e), were designed and synthesized using the previously described ethyl 3-amino-4,9-dioxo-2,3,4,9-tetrahydrothieno[2,3-g]quinoline-3-carboxylate (1) as a starting material. All compounds were evaluated for their antiproliferative activity against a panel of representative liquid and solid human tumor cell lines and exhibit IC₅₀ values in the micromolar/submicromolar range. Series 2 displayed higher cytotoxicity than did series 3. The nature of the substituents on both imidazoline and triazinane N1 nitrogen markedly affected the activity profile of these series. Spectrophotometric and fluorescence measurements as well as unwinding assays performed on the most cytotoxic compounds, 2c, 2g, and 2k, showed that they are nonintercalative DNA agents and inhibit the catalytic activity of Topo II in a concentration-dependent mode. 2g was the most active Topo II inhibitor with activity levels comparable to those of VP-16.

A series of small-molecule Smac mimetics containing a diazabicyclic core structure have been designed, synthesized, and evaluated. The most potent compound (6) binds to XIAP, cIAP-1, and cIAP-2 with K_i values of 8.4, 1.5, and 4.2 nM, respectively, directly antagonizes XIAP in a cell-free functional assay and induces cIAP-1 degradation in cancer cells. It inhibits cell growth with an IC₅₀ value of 31 nM, effectively induces apoptosis in the MDA-MB-231 cancer cell line, and has a good oral bioavailability.

A series of aroylnaphthalene derivatives were prepared as bioisosteres of combrestatin A-4 and evaluated for anticancer activity. 2-Amino-1-aroylnaphthalene and 2-hydroxy-1-aroylnaphthalene, 9 and 8, respectively, showed strong antiproliferative activity with IC₅₀ values of 2.1−26.3 nM against a panel of human cancer cell lines including multiple-drug resistant cell line. Compound 9 demonstrated better antiproliferative activity and has a comparable tubulin binding efficacy as that of colchicine.

Synthesis and characterization of (R)-N^α-(2,2-diphenylacetyl)-N-(4-hydroxybenzyl)-N^ω-([2,3-³H]-propanoyl)argininamide ([³H]-UR-MK114), an easily accessible tritium-labeled NPY Y₁ receptor (Y₁R) antagonist (K_B: 0.8 nM, calcium assay, HEL cells) derived from the (R)-argininamide BIBP 3226, is reported. The radioligand binds with high affinity (K_D, saturation: 1.2 nM, kinetic experiments: 1.1 nM, SK-N-MC cells) and selectivity for Y₁R over Y₂, Y₄, and Y₅ receptors. The title compound is a useful pharmacological tool for the determination of Y₁R ligand affinities, quantification of Y₁R binding sites, and autoradiography.

The recently discovered mammalian molybdoprotein mARC1 is capable of reducing N-hydroxylated compounds. Upon reconstitution with cytochrome b₅ and b₅ reductase, benzamidoxime, pentamidine, and diminazene amidoximes, N-hydroxymelagatran, guanoxabenz, and N-hydroxydebrisoquine are efficiently reduced. These substances are amidoxime/N-hydroxyguanidine prodrugs, leading to improved bioavailability compared to the active amidines/guanidines. Thus, the recombinant enzyme allows prediction about in vivo reduction of N-hydroxylated prodrugs. Furthermore, the prodrug principle is not dependent on cytochrome P450 enzymes.