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Endomorphins (EMs) are potent pharmaceuticals for the treatment of pain. Herein, we investigated several novel EM analogues with multiple modifications and oligoarginine conjugation. Our results showed that analogues 1-6 behaved as potent μ-opioid agonists and enhanced stability and lipophilicity. Analogues 5 and 6 administered centrally and peripherally induced significant and prolonged antinociceptive effects in acute pain. Both analogues also produced long-acting antiallodynic effects against neuropathic and inflammatory pain. Furthermore, they showed a reduced acute antinociceptive tolerance. Analogue 6 decreased the extent of chronic antinociceptive tolerance, and analogue 5 exhibited no tolerance at the supraspinal level. Particularly, they displayed nontolerance-forming antinociception at the peripheral level. In addition, analogues 5 and 6 exhibited reduced or no opioid-like side effects on gastrointestinal transit, conditioned place preference (CPP), and motor impairment. The present investigation established that multiple modifications and oligoarginine-vector conjugation of EMs would be helpful in developing novel analgesics with fewer side effects.

We have designed, synthesized, and evaluated a new class of potent HIV-1 protease inhibitors with novel bicyclic oxazolidinone derivatives as the P2 ligand. We have developed an enantioselective synthesis of these bicyclic oxazolidinones utilizing a key o-iodoxybenzoic acid mediated cyclization. Several inhibitors displayed good to excellent activity toward HIV-1 protease and significant antiviral activity in MT-4 cells. Compound 4k has shown an enzyme K(i) of 40 pM and antiviral IC(50) of 31 nM. Inhibitors 4k and 4l were evaluated against a panel of highly resistant multidrug-resistant HIV-1 variants, and their fold-changes in antiviral activity were similar to those observed with darunavir. Additionally, two X-ray crystal structures of the related inhibitors 4a and 4e bound to HIV-1 protease were determined at 1.22 and 1.30 Å resolution, respectively, and revealed important interactions in the active site that have not yet been explored.

We describe here design, synthesis, and biological evaluation of a series of highly potent HIV-1 protease inhibitors containing stereochemically defined and unprecedented tricyclic furanofuran derivatives as P2 ligands in combination with a variety of sulfonamide derivatives as P2’ ligands. These inhibitors were designed to enhance the ligand-backbone binding and van der Waals interactions in the protease active site. A number of inhibitors containing the new P2 ligand, an aminobenzothiazole as the P2’ ligand and a difluorophenylmethyl as the P1 ligand, displayed very potent enzyme inhibitory potency and also showed excellent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The tricyclic P2 ligand has been synthesized efficiently in an optically active form using enzymatic desymmetrization of meso-1,2-(dihydroxymethyl)cyclohex-4-ene as the key step. We determined high-resolution X-ray structures of inhibitor-bound HIV-1 protease. These structures revealed extensive interactions with the backbone atoms of HIV-1 protease and provided molecular insights into the binding properties of these new inhibitors.

The design, synthesis, and biological evaluation of a new class of HIV-1 protease inhibitors containing stereochemically defined fused tricyclic polyethers as the P2 ligands and a variety of sulfonamide derivatives as the P2′ ligands, are described. A number of ring sizes and various substituent effects were investigated to enhance the ligand-backbone interactions in the protease active site. Inhibitors 5c and 5d containing this unprecedented fused 6-5-5 ring system as the P2 ligand, an aminobenzothiazole as the P2′ ligand and a difluorophenylmethyl as the P1 ligand exhibited exceptional enzyme inhibitory potency and maintained excellent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The umbrella-like P2 ligand for these inhibitors has been synthesized efficiently in an optically active form using a Pauson-Khand cyclization reaction as the key step. The racemic alcohols were resolved efficiently using a lipase catalyzed enzymatic resolution. Two high resolution X-ray structures of inhibitor-bound HIV-1 protease revealed extensive interactions with the backbone atoms of HIV-1 protease and provided molecular insight into the binding properties of these new inhibitors.

In the present study, a series of novel dual-target histone deacetylase (HDAC) and mammalian target of rapamycin (mTOR) inhibitors were designed and synthesized using pyrimidine-pyrazolyl pharmacophore to append HDAC recognition cap and hydroxamic acid as a zinc-binding motif. Among them, 12l was the optimal lead compound with potent inhibition activities against mTOR and HDAC1 with half-maximal inhibitory concentration of 1.2 and 0.19 nM, respectively. Western blot confirmed that 12l could upregulate acetylation of H3 and α-tubulin and downregulate mTOR-related downstream mediators. 12l could also stimulate cell cycle arrest in G0/G1 phase and induce tumor cell apoptosis. 12l showed comparable antitumor activity with the combination medication in MM1S xenograft model with a tumor growth inhibitory rate of 72.5%, without causing significant loss of body weight and toxicity. All of the results indicated that 12l could be a promising dual target inhibitor for treating hematologic malignancies.

In the present study, a series of novel histone deacetylase (HDAC) inhibitors using the morpholinopurine as the capping group were designed and synthesized. Several compounds demonstrated significant HDAC inhibitory activities and antiproliferative effects against diverse human tumor cell lines. Among them, compound 10o was identified as a potent class I and class IIb HDAC inhibitor with good pharmaceutical profile and druglike properties. Western blot analysis further confirmed that 10o more effectively increased acetylated histone H3 than panobinostat (LBH-589) and vorinostat (SAHA) at the same concentration in vitro. In in vivo efficacy evaluations of HCT116, MV4-11, Ramos, and MM1S xenograft models, 10o showed higher efficacy than SAHA or LBH-589 without causing significant loss of body weight and toxicity. All the results indicated that 10o could be a suitable candidate for treatment of both solid and hematological cancer.

The design, synthesis, and biological evaluation of a series of HIV-1 protease inhibitors incorporating stereochemically defined fused tricyclic P2-ligands are described. Various substituent effects were investigated in order to maximize the ligand-binding site interactions in the protease active site. Inhibitors 16a and 16f showed excellent enzyme inhibitory and antiviral activity while incorporation of sulfone functionality resulted in a decrease in potency. Both inhibitors 16a and 16f have maintained activity against a panel of multidrug resistant HIV-1 variants. A high-resolution X-ray crystal structure of 16a-bound HIV-1 protease revealed important molecular insights into the ligand-binding site interactions which may account for the inhibitor’s potent antiviral activity and excellent resistance profiles.

The mitotic kinesin Eg5 is critical for the assembly of the mitotic spindle and is a promising chemotherapy target. Previously, we identified S-trityl-L-cysteine as a selective inhibitor of Eg5 and developed triphenylbutanamine analogues with improved potency, favorable drug-like properties, but moderate in vivo activity. We report here their further optimization to produce extremely potent inhibitors of Eg5 (K(i)(app)10 nM) with broad-spectrum activity against cancer cell lines comparable to the Phase II drug candidates ispinesib and SB-743921. They have good oral bioavailability and pharmacokinetics and induced complete tumor regression in nude mice explanted with lung cancer patient xenografts. Furthermore, they display fewer liabilities with CYP-metabolizing enzymes and hERG compared with ispinesib and SB-743921, which is important given the likely application of Eg5 inhibitors in combination therapies. We present the case for this preclinical series to be investigated in single and combination chemotherapies, especially targeting hematological malignancies.

A series of potent HIV-1 protease inhibitors with a lipophilic adamantyl P1 ligand have been designed, synthesized, and evaluated. We have developed an enantioselective synthesis of adamantane-derived hydroxyethylamine isosteres utilizing Sharpless asymmetric epoxidation as the key step. Various inhibitors incorporating P1-adamantylmethyl in combination with P2 ligands such as 3-(R)-THF, 3-(S)-THF, bis-THF, and THF-THP were examined. The S1′ pocket was also probed with phenyl and phenylmethyl ligands. Inhibitor 15d, with an isobutyl P1′ ligand and a bis-THF P2 ligand, proved to be the most potent of the series. The cLogP value of inhibitor 15d is improved compared to inhibitor 2 with a phenylmethyl P1-ligand. X-ray structural studies of 15d, 15h, and 15i with HIV-1 protease complexes revealed molecular insight into the inhibitor–protein interaction.

Cooperating mercapto groups with tacrine in a single molecular, novel multifunctional compounds have been designed and synthesized. These mercapto-tacrine derivatives displayed a synergistic pharmacological profile of long-term potentiation enhancement, cholinesterase inhibition, neuroprotection, and less hepatotoxicity, emerging as promising molecules for the therapy of age-related neurodegenerative diseases.

The human mitotic kinesin Eg5 represents a novel mitotic spindle target for cancer chemotherapy. We previously identified S-trityl-l-cysteine (STLC) and related analogues as selective potent inhibitors of Eg5. We herein report on the development of a series of 4,4,4-triphenylbutan-1-amine inhibitors derived from the STLC scaffold. This new generation systematically improves on potency: the most potent C-trityl analogues exhibit K(i)(app) ≤ 10 nM and GI(50) ≈ 50 nM, comparable to results from the phase II clinical benchmark ispinesib. Crystallographic studies reveal that they adopt the same overall binding configuration as S-trityl analogues at an allosteric site formed by loop L5 of Eg5. Evaluation of their druglike properties reveals favorable profiles for future development and, in the clinical candidate ispinesib, moderate hERG and CYP inhibition. One triphenylbutanamine analogue and ispinesib possess very good bioavailability (51% and 45%, respectively), with the former showing in vivo antitumor growth activity in nude mice xenograft studies.

Novel selective histone deacetylase 6 (HDAC6) inhibitors using the quinazoline as the cap were designed, synthesized, and evaluated for HDAC enzymatic assays. N-Hydroxy-4-(2-methoxy-5-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)butanamide, 23bb, was the most potent selective inhibitor for HDAC6 with an IC50 of 17 nM and showed 25-fold and 200-fold selectivity relative to HDAC1 and HDAC8, respectively. In vitro, 23bb presented low nanomolar antiproliferative effects against panel of cancer cell lines. Western blot analysis further confirmed that 23bb increased acetylation level of α-tubulin in vitro. 23bb has a good pharmacokinetic profile with oral bioavailability of 47.0% in rats. In in vivo efficacy evaluations of colorectal HCT116, acute myelocytic leukemia MV4-11, and B cell lymphoma Romas xenografts, 23bb more effectively inhibited the tumor growth than SAHA even at a 4-fold reduced dose or ACY-1215 at the same dose. Our results indicated that 23bb is a potent oral anticancer candidate for selective HDAC6 inhibitor and deserves further investigation.

We report the design, synthesis, biological evaluation, and the X-ray crystal structure of a novel inhibitor bound to the HIV-1 protease. Various C3-functionalized cyclopentanyltetrahydrofurans (Cp-THF) were designed to interact with the flap Gly48 carbonyl or amide NH in the S2-subsite of the HIV-1 protease. We investigated the potential of those functionalized ligands in combination with hydroxyethylsulfonamide isosteres. Inhibitor 26 containing a 3-(R)-hydroxyl group on the Cp-THF core displayed the most potent enzyme inhibitory and antiviral activity. Our studies revealed a preference for the 3-(R)-configuration over the corresponding 3-(S)-derivative. Inhibitor 26 exhibited potent activity against a panel of multidrug-resistant HIV-1 variants. A high resolution X-ray structure of 26-bound HIV-1 protease revealed important molecular insight into the ligand-binding site interactions.

The structure-based design, synthesis, and biological evaluation of a series of nonpeptidic macrocyclic HIV protease inhibitors are described. The inhibitors are designed to effectively fill in the hydrophobic pocket in the S1'-S2' subsites and retain all major hydrogen bonding interactions with the protein backbone similar to darunavir (1) or inhibitor 2. The ring size, the effect of methyl substitution, and unsaturation within the macrocyclic ring structure were assessed. In general, cyclic inhibitors were significantly more potent than their acyclic homologues, saturated rings were less active than their unsaturated analogues and a preference for 10- and 13-membered macrocylic rings was revealed. The addition of methyl substituents resulted in a reduction of potency. Both inhibitors 14b and 14c exhibited marked enzyme inhibitory and antiviral activity, and they exerted potent activity against multidrug-resistant HIV-1 variants. Protein-ligand X-ray structures of inhibitors 2 and 14c provided critical molecular insights into the ligand-binding site interactions.

Structure-based design, synthesis, and biological evaluation of a series of novel HIV-1 protease inhibitors are described. In an effort to enhance interactions with protease backbone atoms, we have incorporated stereochemically defined methyl-2-pyrrolidinone and methyl oxazolidinone as the P1'-ligands. These ligands are designed to interact with Gly-27' carbonyl and Arg-8 side chain in the S1'-subsite of the HIV protease. We have investigated the potential of these ligands in combination with our previously developed bis-tetrahydrofuran (bis-THF) and cyclopentanyltetrahydrofuran (Cp-THF) as the P2-ligands. Inhibitor 19b with a (R)-aminomethyl-2-pyrrolidinone and a Cp-THF was shown to be the most potent compound. This inhibitor maintained near full potency against multi-PI-resistant clinical HIV-1 variants. A high resolution protein-ligand X-ray crystal structure of 19b-bound HIV-1 protease revealed that the P1'-pyrrolidinone heterocycle and the P2-Cp-ligand are involved in several critical interactions with the backbone atoms in the S1' and S2 subsites of HIV-1 protease.

We report the design, synthesis and biological evaluation of a series of novel HIV-1 protease inhibitors. The inhibitors incorporate stereochemically defined flexible cyclic ethers/polyethers as the high affinity P2-ligands. Inhibitors containing small ring 1,3-dioxacycloalkanes have shown potent enzyme inhibitory and antiviral activity. Inhibitors 3d and 3h are the most active inhibitors. Inhibitor 3d maintains excellent potency against a variety of multi-PI-resistant clinical strains. Our structure-activity studies indicate that the ring size, stereochemistry, and position of oxygens are important for the observed activity. Optically active synthesis of 1,3-dioxepan-5-ol along with the syntheses of various cyclic ether and polyether ligands have been described. A protein-ligand X-ray crystal structure of 3d-bound HIV-1 protease was determined. The structure revealed that the P2-ligand makes extensive interactions including hydrogen bonding with the protease backbone in the S2-site. In addition, the P2-ligand in 3d forms a unique water-mediated interaction with the NH of Gly-48.

Structure-based design, synthesis, and biological evaluation of a series of very potent HIV-1 protease inhibitors are described. In an effort to improve backbone ligand-binding site interactions, we have incorporated basic-amines at the C4 position of the bis-tetrahydrofuran (bis-THF) ring. We speculated that these substituents would make hydrogen bonding interactions in the flap region of HIV-1 protease. Synthesis of these inhibitors was performed diastereoselectively. A number of inhibitors displayed very potent enzyme inhibitory and antiviral activity. Inhibitors 25f, 25i, and 25j were evaluated against a number of highly-PI-resistant HIV-1 strains, and they exhibited improved antiviral activity over darunavir. Two high resolution X-ray structures of 25f- and 25g-bound HIV-1 protease revealed unique hydrogen bonding interactions with the backbone carbonyl group of Gly48 as well as with the backbone NH of Gly48 in the flap region of the enzyme active site. These ligand-binding site interactions are possibly responsible for their potent activity.

We report the design, synthesis, X-ray structural studies, and biological evaluation of a novel series of HIV-1 protease inhibitors. We designed a variety of functionalized biphenyl derivatives to make enhanced van der Waals interactions in the S1 subsite of HIV-1 protease. These biphenyl derivatives were conveniently synthesized using a Suzuki-Miyaura cross-coupling reaction as the key step. We examined the potential of these functionalized biphenyl-derived P1 ligands in combination with 3-(S)-tetrahydrofuranyl urethane and bis-tetrahydrofuranyl urethane as the P2 ligands. Inhibitor 21e, with a 2-methoxy-1, 1’-biphenyl derivative as P1 ligand and bis-THF as the P2 ligand, displayed the most potent enzyme inhibitory and antiviral activity. This inhibitor also exhibited potent activity against a panel of multidrug-resistant HIV-1 variants. A high resolution X-ray crystal structure of related Boc-derivative 17a-bound HIV-1 protease provided important molecular insight into the ligand-binding site interactions of the biphenyl core in the S1 subsite of HIV-1 protease.

The potent new antiviral inhibitor TMC-114 (UIC-94017) of HIV-1 protease (PR) has been studied with three PR variants containing single mutations D30N, I50V, and L90M, which provide resistance to the major clinical inhibitors. The inhibition constants (K(i)) of TMC-114 for mutants PR(D30N), PR(I50V), and PR(L90M) were 30-, 9-, and 0.14-fold, respectively, relative to wild-type PR. The molecular basis for the inhibition was analyzed using high-resolution (1.22-1.45 A) crystal structures of PR mutant complexes with TMC-114. In PR(D30N), the inhibitor has a water-mediated interaction with the side chain of Asn30 rather than the direct interaction observed in PR, which is consistent with the relative inhibition. Similarly, in PR(I50V) the inhibitor loses favorable hydrophobic interactions with the side chain of Val50. TMC-114 has additional van der Waals contacts in PR(L90M) structure compared to the PR structure, leading to a tighter binding of the inhibitor. The observed changes in PR structure and activity are discussed in relation to the potential for development of resistant mutants on exposure to TMC-114.

Twenty-one novel derivatives of millepachine were synthesized and evaluated for their in vitro antiproliferative activity. Among them, 8 exhibited the most potent activity, with IC50 values of 8-27 nM against panel of cancer cell lines and retained full activity in multidrug resistant cancer cells. Treated cells were arrested in G2/M phase and resulted in cellular apoptosis. Microtubule dynamics confirmed 8 was a novel tubulin polymerization inhibitor by binding at the colchicine site. 8 also exhibited antivascular activity because it concentration dependently reduced the cell migration and disrupted capillary like tube formation in HUVEC cells. Furthermore, the hydrochloride salt of 8 (8·HCl) significantly improved the bioavailability up to 47% while retaining the antiproliferative activity. Importantly, 8·HCl significantly inhibited tumor growths in four xenograft models including resistance tumor-cell-bearing mice models without causing significant loss of body weight, suggesting that 8 is a promising new orally anticancer agent to be developed.

Extreme drug resistant mutant of HIV-1 protease (PR) bearing 20 mutations (PR20) has been studied with the clinical inhibitor amprenavir (1) and two potent antiviral investigational inhibitors GRL-02031 (2) and GRL-0519 (3). Clinical inhibitors are >1000-fold less active on PR20 than on wild-type enzyme, which is consistent with dissociation constants (KL) from isothermal titration calorimetry of 40 nM for 3, 178 nM for amprenavir, and 960 nM for 2. High resolution crystal structures of PR20-inhibitor complexes revealed altered interactions compared with the corresponding wild-type PR complexes in agreement with relative inhibition. Amprenavir lacks interactions due to PR20 mutations in the S2/S2' subsites relative to PR. Inhibitors 2 and 3 lose interactions with Arg8' in PR20 relative to the wild-type enzyme because Arg8' shifts to interact with mutated L10F side chain. Overall, inhibitor 3 compares favorably with darunavir in affinity for PR20 and shows promise for further development.

GRL-0519 (1) is a potent antiviral inhibitor of HIV-1 protease (PR) possessing tris-tetrahydrofuran (tris-THF) at P2. The high resolution X-ray crystal structures of inhibitor 1 in complexes with single substitution mutants PR(R8Q), PR(D30N), PR(I50V), PR(I54M), and PR(V82A) were analyzed in relation to kinetic data. The smaller valine side chain in PR(I50V) eliminated hydrophobic interactions with inhibitor and the other subunit consistent with 60-fold worse inhibition. Asn30 in PR(D30N) showed altered interactions with neighboring residues and 18-fold worse inhibition. Mutations V82A and I54M showed compensating structural changes consistent with 6-7-fold lower inhibition. Gln8 in PR(R8Q) replaced the ionic interactions of wild type Arg8 with hydrogen bond interactions without changing the inhibition significantly. The carbonyl oxygen of Gly48 showed two alternative conformations in all structures likely due to the snug fit of the large tris-THF group in the S2 subsite in agreement with high antiviral efficacy of 1 on resistant virus.

GRL-02031 (1) is an HIV-1 protease (PR) inhibitor containing a novel P1' (R)-aminomethyl-2-pyrrolidinone group. Crystal structures at resolutions of 1.25-1.55 A were analyzed for complexes of 1 with the PR containing major drug resistant mutations, PR(I47V), PR(L76V), PR(V82A), and PR(N88D). Mutations of I47V and V82A alter residues in the inhibitor-binding site, while L76V and N88D are distal mutations having no direct contact with the inhibitor. Substitution of a smaller amino acid in PR(I47V) and PR(L76V) and the altered charge of PR(N88D) are associated with significant local structural changes compared to the wild-type PR(WT), while substitution of alanine in PR(V82A) increases the size of the S1' subsite. The P1' pyrrolidinone group of 1 accommodates to these local changes by assuming two different conformations. Overall, the conformation and interactions of 1 with PR mutants resemble those of PR(WT) with similar inhibition constants in good agreement with the antiviral potency on multidrug resistant HIV-1.

Virtual screening targeting the urokinase receptor (uPAR) led to (3R)-4-cyclohexyl-3-(hexahydrobenzo[d][1,3]dioxol-5-yl)-N-((hexahydrobenzo[d][1,3]dioxol-5-yl)methyl)butan-1-aminium 1 (IPR-1) and 4-(4-((3,5-dimethylcyclohexyl)carbamoyl)-2-(4-isopropylcyclohexyl)pyrazolidin-3-yl)piperidin-1-ium 3 (IPR-69). Synthesis of an analog of 1, namely 2 (IPR-9), and 3 led to breast MDA-MB-231 invasion, migration and adhesion assays with IC50 near 30 μM. Both compounds blocked angiogenesis with IC50 of 3 μM. Compounds 2 and 3 inhibited cell growth with IC50 of 6 and 18 μM and induced apoptosis. Biochemical assays revealed lead-like properties for 3, but not 2. Compound 3 administered orally reached peak concentration of nearly 40 μM with a half-life of about 2 hours. In NOD-SCID mice inoculated with breast TMD-231 cells in their mammary fat pads, compound 3 showed a 20% reduction in tumor volumes and less extensive metastasis was observed for the treated mice. The suitable pharmacokinetic properties of 3 and the encouraging preliminary results in metastasis make it an ideal starting point for next generation compounds.

The potent new antiviral inhibitor GRL-98065 (1) of HIV-1 protease (PR) has been studied with PR variants containing the single mutations D30N, I50V, V82A, and I84V that provide resistance to the major clinical inhibitors. Compound 1 had inhibition constants of 17-fold, 8-fold, 3-fold, and 3-fold, respectively, for PR(D30N), PR(I50V), PR(V82A), and PR(I84V) relative to wild type PR. The chemically related darunavir had similar relative inhibition, except for PR(D30N), where inhibitor 1 was approximately 3-fold less potent. The high resolution (1.11-1.60 Angstrom) crystal structures of PR mutant complexes with inhibitor 1 showed small changes relative to the wild type enzyme. PR(D30N) and PR(V82A) showed compensating interactions with inhibitor 1 relative to those of PR, while reduced hydrophobic contacts were observed with PR(I50V) and PR(I84V). Importantly, inhibitor 1 complexes showed fewer changes relative to wild type enzyme than reported for darunavir complexes. Therefore, inhibitor 1 is a valuable addition to the antiviral inhibitors with high potency against resistant strains of HIV.