A series of polyaminohydroxamic acids (PAHAs) and polyaminobenzamides (PABAs) were synthesized and evaluated as isoform-selective histone deacetylase (HDAC) inhibitors. These analogues contain a polyamine chain to increase affinity for chromatin and facilitate cellular import. Seven PAHAs inhibited HDAC >50% (1 µM), and two PABAs inhibited HDAC >50% (5 µM). Compound 17 increased acetylated α-tubulin in HCT116 colon tumor cells 253-fold but only modestly increased p21waf1 and acetylated histones 3 and 4, suggesting that 17 selectively inhibits HDAC 6. PABA 22 alone minimally increased p21waf1 and acetylated histones 3 and 4 but caused dose-dependent increases in p21waf1 in combination with 0.1 µM 5-azadeoxycytidine. Finally, 22 appeared to be a substrate for the polyamine transport system. None of these compounds were cytotoxic at 100 µM. PAHAs and PABAs exhibit strikingly different cellular effects from SAHA and have the potential for use in combination antitumor therapies with reduced toxicity.

Histone deacetylase inhibitors (HDACi) are a relatively new class of chemotherapy agents. Herein, we report a click-chemistry based approach to the synthesis of HDACi. Fourteen agents were synthesized from the combination of two alkyne and seven azido precursors. The inhibition of HDAC1 and HDAC8 was then determined by in vitro enzymatic assays, after which the cytotoxicity was evaluated in the NCI human cancer cell line screen. A lead compound 5g (NSC746457) was discovered that inhibited HDAC1 at an IC50 value of 104 ± 30 nM and proved quite potent in the cancer cell line screen with GI50 values ranging from 3.92 μM to 10 nM. Thus, this click HDACi design has provided a new chemical scaffold that has not only revealed a lead compound, but one which is easily amendable to further structural modifications given the modular nature of this approach.

The phenylethylene diamines are a class of σ receptor ligands with excellent selectivity over other biological systems and with anti-cocaine actions that involve antagonism of σ1 receptors. In order to increase the potency of the aromatic methoxyl substituted analogues, trifluoromethoxyl groups were introduced to prevent metabolic demethylation. The para-substituted trifluoromethoxyl substituted analogues were shown to have increased σ receptor affinity and represent the most potent anti-cocaine phenylethylene diamines yet described.

Continuing our studies investigating opioids with reduced P-glycoprotein (P-gp) substrate activity, a series of known 3- and 6-hydroxy, -methoxy, and -desoxymorphine analogs was synthesized and analyzed for P-gp substrate activity and opioid binding affinity. 6-Desoxymorphine (7) showed high affinity for opioid receptors and did not induce P-gp-mediated ATP hydrolysis. Additionally, 7 demonstrated morphine-like antinociceptive potency in mice, indicating this compound as an ideal lead to further evaluate the role of P-gp in opioid analgesic tolerance development.

The prostate-specific membrane antigen (PSMA) is increasingly recognized as a viable target for imaging and therapy of cancer. We prepared seven 99mTc/Re-labeled compounds by attaching known Tc/Re chelating agents to an amino-functionalized PSMA inhibitor (lys-NHCONH-glu) with or without a variable length linker moiety. Ki values ranged from 0.17 to 199 nM. Ex vivo biodistribution and in vivo imaging demonstrated the degree of specific binding to engineered PSMA+ PC3 PIP tumors. PC3-PIP cells are derived from PC3 that have been transduced with the gene for PSMA. Despite demonstrating nearly the lowest PSMA inhibitory potency of this series, [99mTc(CO)3(L1)]+ (L1 = (2-pyridylmethyl)2N(CH2)4CH(CO2H)-NHCO-(CH2)6CO-NH-lys-NHCONH-glu) showed the highest, most selective PIP tumor uptake, at 7.9 ± 4.0% injected dose per gram of tissue at 30 min postinjection. Radioactivity cleared from nontarget tissues to produce a PIP to flu (PSMA-PC3) ratio of 44:1 at 120 min postinjection. PSMA can accommodate the steric requirements of 99mTc/Re complexes within PSMA inhibitors, the best results achieved with a linker moiety between the ε amine of the urea lysine and the chelator.

Fatty acid synthase (FAS) is necessary for growth and survival of tumor cells and is a promising drug target for oncology. Here, we report on the syntheses and activity of novel inhibitors of the thioesterase domain of FAS. Using the structure of orlistat as a starting point, which contains a β-lactone as the central pharmacophore, 28 novel congeners were synthesized and examined. Structural features such as the length of the α- and β-alkyl chains, their chemical composition, and amino ester substitutions were altered and the resulting compounds explored for inhibitory activity toward the thioesterase domain of FAS. Nineteen congeners show improved potency for FAS in biochemical assays relative to orlistat. Three of that subset, including the natural product valilactone, also display an increased potency in inducing tumor cell death and improved solubility compared to orlistat. These findings support the idea that an orlistat congener can be optimized for use in a preclinical drug design and for clinical drug development.

N-(5-(2-(5-Chloro-2-methoxyphenylamino)thiazol-4-yl)-4-methylthiazol-2-yl)pivalamide 1 (compound 15Jf) was found previously to correct defective cellular processing of the cystic fibrosis protein ΔF508-CFTR. Eight C4′-C5 C,C-bond-controlling bithiazole analogs of 1 were designed, synthesized, and evaluated to establish that constraining rotation about the bithiazole-tethering has a significant effect on corrector activity. For example, constraining the C4′-C5 bithiazole tether in the s-cis conformation [N-(2-(5-chloro-2-methoxyphenyl-amino)-7,8-dihydro-6H-cyclohepta[1,2-d:3,4-d′]bithiazole-2′-yl)pivalamide; 29] results in improved corrector activity. Heteroatom placement in the bithaizole core is also critical as evidenced by the decisive loss of corrector activity with s-cis constrained N-(2-(5-chloro-2-methoxyphenylamino)-5,6-dihydro-4H-cyclohepta[1,2-d:3,4-d′]bithiazole-2′-yl)pivalamide 33. In addition, computational models were utilized to examine the conformational preferences for select model systems. Following our analysis, the “s-cis locked” cycloheptathiazolothiazole 29 was found to be the most potent bithiazole corrector, with an IC50 of ~450 nM.

Indoleamine 2,3-dioxygenase (IDO) is emerging as an important new therapeutic target for the treatment of cancer, chronic viral infections, and other diseases characterized by pathological immune suppression. With the goal of developing more potent IDO inhibitors, a systematic study of 4-phenyl-imidazole (4-PI) derivatives was undertaken. Computational docking experiments guided design and synthesis efforts with analogs of 4-PI. In particular, three interactions of 4-PI analogs with IDO were studied: the active site entrance, the interior of the active site and the heme iron binding. The three most potent inhibitors (1, 17 and 18) appear to exploit interactions with C129 and S167 in the interior of the active site. All three inhibitors are approximately ten-fold more potent than 4-PI. The study represents the first example of enzyme inhibitor development with the recently reported crystal structure of IDO and offers important lessons in the search for more potent inhibitors.

Discovering dopamine D2-like receptor subtype-selective ligands has been a focus of significant investigation. The D2R-selective antagonist 3-[4-(4-chlorophenyl)-4-hydroxypiperidinyl]methylindole (1, L741,626; Ki(D2R/D3R) = 11.2:163 nM) has previously provided a lead template for chemical modification. Herein, analogues have been synthesized where the piperidine was replaced by a tropane ring that reversed the selectivity seen in the parent compound, in human hD2LR- or hD3R-transfected HEK 293 cells (31, Ki(D2R/D3R) = 33.4: 15.5 nM). Further exploration of both N-substituted and aryl ring-substituted analogues resulted in the discovery of several high affinity D2R/D3R ligands with 3-benzofurylmethyl-substituents (e.g., 45, Ki(D2R/D3R) = 1.7:0.34 nM) that induced high affinity not achieved in similarly N-substituted piperidine analogues and significantly (470-fold) improved D3R binding affinity compared to the parent ligand 1. X-ray crystallographic data revealed a distinctive spatial arrangement of pharmacophoric elements in the piperidinol vs tropine analogues, providing clues for the diversity in SAR at the D2 and D3 receptor subtypes.

Enantiomeric propanolamines have been identified as a new class of NR2B-selective NMDA receptor antagonists. The most effective agents are biaryl structures, synthesized in six steps with overall yields ranging from 11–64%. The compounds are potent and selective inhibitors of NR2B-containing recombinant NMDA receptors with IC50 values between 30–100 nM. Potency is strongly controlled by substitution on both rings and the centrally located amine nitrogen. SAR analysis suggests that well-balanced polarity and chain-length factors provide the greatest inhibitory potency. Structural comparisons based on 3D shape analysis and electrostatic complementarity support this conclusion. The antagonists are neuroprotective in both in vitro and in vivo models of ischemic cell death. In addition, some compounds exhibit anticonvulsant properties. Unlike earlier generation NMDA receptor antagonists and some NR2B-selective antagonists, the present series of propanolamines does not cause increased locomotion in rodents. Thus, the NR2B-selective antagonists exhibit a range of therapeutically interesting properties.

Pyrano[3,2-c]pyridone and pyrano[3,2-c]quinolone structural motifs are commonly found in alkaloids manifesting diverse biological activities. As part of a program aimed at structural simplification of bioactive natural products utilizing multicomponent synthetic processes, we developed compound libraries based on these privileged heterocyclic scaffolds. The selected library members display low nanomolar antiproliferative activity and induce apoptosis in human cancer cell lines. Mechanistic studies reveal that these compounds induce cell cycle arrest in the G2/M phase and block in vitro tubulin polymerization. Because of the successful clinical use of microtubule-targeting agents, these heterocyclic libraries are expected to provide promising new leads in anticancer drug design.

The effects of 3-position substitution of 9-aminomethyl-9,10-dihydroanthracene (AMDA) on 5-HT2A receptor affinity were determined and compared to a parallel series of DOB-like 1-(2,5-dimethoxyphenyl)-2-aminopropanes substituted at the 4-position. The results were interpreted within the context of 5-HT2A receptor models that suggest that members of the DOB-like series can bind to the receptor in two distinct modes that correlate with the compounds’ functional activity. Automated ligand docking and molecular dynamics suggest that all of the AMDA derivatives, the parent of which is a 5-HT2A antagonist, bind in a fashion analogous to that for the sterically demanding antagonist DOB-like compounds. The failure of the F3406.52L mutation to adversely affect the affinity of AMDA and the 3-bromo derivative is consistent with the proposed modes of orientation. Evaluation of ligand-receptor complex models suggest that a valine/threonine exchange between the 5-HT2A and D2 receptors may be the origin of selectivity for AMDA and two substituted derivatives.

In mammals, the second messenger cAMP is synthesized by a family of transmembrane isoforms (tmACs) and one known cytoplasmic enzyme, “soluble” adenylyl cyclase (sAC). Understanding the individual contributions of these families to cAMP signaling requires tools which can distinguish them. Here, we describe the structure-based development of isoform discriminating AC inhibitors. Docking calculations using a library of small molecules with the crystal structure of a sAC homologue complexed with the noncompetitive inhibitor catechol estrogen identified two novel inhibitors, 3,20-dioxopregn-4-en-21-yl 4-bromobenzenesulfonate (2) and 1,2,3,4,5,6,7,8,13,13,14,14-dodecachloro-1,4,4a,4b,5,8,8a,12b-octahydro-11-sulfo-1,4:5,8-dimethanotriphenylene-10-carboxylic acid (3). In vitro testing revealed that 3 defines a novel AC inhibitor scaffold with high affinity for human sAC and less inhibitory effect on mammalian tmACs. 2 also discriminates between sAC and tmACs, and it appears to simultaneously block the original binding pocket and a neighboring interaction site. Our results show that compounds exploiting the catechol estrogen binding site can produce potent, isoform discriminating AC inhibitors.

Extensive molecular docking, molecular dynamics simulations, and binding free energy calculations have been performed to understand how α7-specific agonists of nicotinic acetylcholine receptor (nAChR), including AR-R17779 (1), GTS-21 (4), and 4-OH-GTS-21 (5), interact with the α7 receptor, leading to important new insights into the receptor–agonist binding. In particular, the cationic head of 4 and 5 has favorable hydrogen bonding and cation–π interactions with residue Trp149. The computational results have also led us to better understand the roles of Gln117 and other residues in the receptor binding with agonists. The computational predictions are supported by data obtained from wet experimental tests. The new insights into the binding and structure–activity relationship obtained from this study should be valuable for future rational design of more potent and selective agonists of the α7 receptor.

New chemotherapeutics are urgently needed to combat malaria. We previously reported on a novel series of antimalarial, ethylenediamine-based inhibitors of protein farnesyltransferase (PFT). In the current study, we designed and synthesized a series of second generation inhibitors, wherein the core ethylenediamine scaffold was varied in order to examine both the homology model of Plasmodium falciparum PFT (PfPFT) and our predicted inhibitor binding mode. We identified several PfPFT inhibitors (PfPFTIs) that are selective for PfPFT versus the mammalian isoform of the enzyme (up to 136-fold selectivity), that inhibit the malarial enzyme with IC50 values down to 1 nM, and that block the growth of P. falciparum in infected whole cells (erythrocytes) with ED50 values down to 55 nM. The structure–activity data for these second generation, ethylenediamine-inspired PFT inhibitors were rationalized by consideration of the X-ray crystal structure of mammalian PFT and the homology model of the malarial enzyme.

We report a series of inhibitors of secreted phospholipases A2 (sPLA2s) based on substituted indoles, 6,7-benzoindoles, and indolizines derived from LY315920, a well-known indole-based sPLA2 inhibitor. Using the human group X sPLA2 crystal structure, we prepared a highly potent and selective indole-based inhibitor of this enzyme. Also, we report human and mouse group IIA and IIE specific inhibitors and a substituted 6,7-benzoindole that inhibits nearly all human and mouse sPLA2s in the low nanomolar range.

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.

AMPA receptors mediate fast excitatory synaptic transmission and are essential for synaptic plasticity. ANQX, a photoreactive AMPA receptor antagonist, is an important biological probe used to irreversibly inactivate AMPA receptors. Here, using X-ray crystallography and mass spectroscopy, we report that ANQX forms two major products in the presence of the GluR2 AMPAR ligand-binding core (S1S2J). Upon photostimulation, ANQX reacts intramolecularly to form FQX or intermolecularly to form a covalent adduct with Glu705.

Structure-activity correlations were investigated for substituted peptide conjugates that function as dual receptor site antagonists of HIV-1 gp120. A series of peptide conjugates were constructed via click reaction of both aryl and alkyl acetylenes with an internally-incorporated azidoproline 6 derived from the parent peptide 1 (12p1, RINNIPWSEAMM). Compared to 1, many of these conjugates were found to exhibit several orders of magnitude increase in both affinity for HIV-1 gp120 and inhibition potencies at both the CD4 and co-receptor binding sites of gp120. We sought to determine structural factors in the added triazole grouping responsible for the increased binding affinity and antiviral activity of the dual inhibitor conjugates. We measured peptide conjugate potencies in both kinetic and cell infection assays. High affinity was sterically specific, being exhibited by the cis but not the trans triazole. The results demonstrate that aromatic, hydrophobic and steric features in the residue 6 side-chain are important for increased affinity and inhibition. Optimizing these features provides a basis for developing gp120 dual inhibitors into peptidomimetic and increasingly smaller molecular weight entry antagonist leads.

The 2’-deoxynucleoside 5’-triphosphate (dNTP) analogs with modifications in the triphosphate chain have been used as nucleotide probes in various biochemical and structural studies. Here we report synthesis and characterization of a complete set of α,β-methylene-2’-dNTPs (α,β-m-dNTP; N = A, C, T, G, 12-15), in which the α,β-oxygen linkage of natural dNTP was replaced isosterically by a methylene group. These nucleotides were designed to be non-cleavable DNA polymerase substrates. Our synthesis process entails preparation of 2’-deoxynucleoside 5’-diphosphate precursors by nucleophilic coupling of 5’-tosyl nucleosides and methylene-diphosphate, and a subsequent enzymatic γ-phosphorylation. All four synthesized α,β-m-dNTPs were found to be potent inhibitors of polymerase β with Ki values ranging from 1-5 μM. During preparation of the dG and dT derivatives of α,β-methylene diphosphate, we isolated significant amounts of 3,5’-anhydro-2’-deoxyguanosine (cyclo-dG, 16) and 2,5’-anhydro-2’-deoxythymidine (cyclo-dT, 17), respectively. These novel 2’-deoxycyclonucleosides were formed via a base-catalyzed deprotonation of the imino proton (N1-H and N3-H), followed by an intramolecular cyclization (N3 → C5’ and O2 → C5’, respectively). In acidic solution, the cyclonucleosides underwent glycolysis at N9, followed by complete depurination at N3. In the case of cyclo-dG, there existed an equilibrium between glycolysis and deglycolysis at the glycosidic linkage prior to complete depurination. When exposed to alkaline conditions, cyclo-dG underwent an oxidative deamination at C2 to produce 3,5’-anhydro-2’-deoxyxanthosine (cyclo-dX, 19), whereas cyclo-dT was hydrolyzed exclusively to dT via cleavage at the 2,5’-ether linkage.

The melanocortin-3 and -4 receptors (MC3R, MC4R) have been implicated in energy homeostasis and obesity. Whereas the physiological role of the MC4R is extensively studied, little is known about the MC3R. One caveat is the limited availability of ligands that are selective for the MC3R. Previous studies identified Ac-His-DPhe(p-I)-Arg-Trp-NH2, which possessed partial agonist/antagonist pharmacology at the mMC3R while retaining full nanomolar agonist pharmacology at the mMC4R. These data allowed for the hypothesis that the DPhe position in melanocortin tetrapeptides can be used to examine ligand side-chain determinants important for differentiation of mMC3R agonist versus antagonist activity. A series of 15 DPhe7 modified Ac-His-DPhe7-Arg-Trp-NH2 tetrapeptides has been synthesized and pharmacologically characterized. Most notable results include the identification of modifications that resulted in potent antagonists/partial agonists at the mMC3R and full, potent agonists at the mMC4R. These SAR studies provide experimental evidence that the molecular mechanism of antagonism at the mMC3R differentiates this subtype from the mMC4R.

MbtA (a salicyl AMP ligase) is a key target for the design of new antitubercular agents. Based on structure activity relationship (SAR) data generated in our laboratory, a structure-based model is developed to predict the binding affinities of arylacid-AMP bisubstrate inhibitors of MbtA. The approach described takes advantage of the linear interaction energy (LIE) technique to derive linear equations relating ligand structure to function. Using only two parameters derived from molecular dynamics simulations good correlation (R2 = 0.70) was achieved for a set of 31 inhibitors with binding affinities spanning six orders of magnitude. The results were applied to understand the effect of steric and heteroatom substitutions on bisubstrate ligand binding and to predict second generation inhibitors of MbtA. The resulting model was further validated by chemical synthesis of a novel inhibitor with a predicted LIE binding affinity of 1.6 nM and a subsequently determined experimental Kiapp of 0.7 nM.

Gedunin (1), a tetranortriterpenoid isolated from the Indian neem tree (Azadirachta indica), was recently shown to manifest anticancer activity via inhibition of the 90 kDa heat shock protein (Hsp90) folding machinery and to induce the degradation of Hsp90-dependent client proteins similar to other Hsp90 inhibitors. The mechanism of action by which gedunin induces client protein degradation remains undetermined, however, prior studies have demonstrated that it does not bind competitively versus ATP. In an effort to further probe the mechanism of action, 19 semisynthetic derivatives of gedunin were prepared and their antiproliferative activity against MCF-7 and SkBr3 breast cancer cells determined. Although no compound was found to exhibit antiproliferative activity more effective than the natural product, functionalities critical for antiproliferative activity have been identified.

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 Ki values of 3.5 nM 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.

We report on the synthesis and evaluation of an indazole-spin-labeled compound that was designed as an effective chemical probe for second site screening against the protein kinase JNK using NMR-based techniques. We demonstrate the utility of the derived compound in detecting and characterizing binding events at the protein kinase docking site. In addition, we report on the NMR-based design and synthesis of a bidentate compound spanning both the ATP site and the docking site. We show that the resulting compound has nanomolar affinity for JNK despite the relatively weak affinities of the individual fragments that constitute it. The approach demonstrates that targeting the docking site of protein kinases represents a valuable yet unexplored avenue to obtain potent kinase inhibitors with increased selectivity.