Tyrosine-sulfate isosteres of CCR5 N-terminus as tools for studying HIV-1 entry

The HIV-1 co-receptor CCR5 possesses sulfo-tyrosine (TYS) residues at its N-terminus (Nt) that are required for binding HIV-1 gp120 and mediating viral entry. By using a 14-residue fragment of CCR5 Nt containing two TYS residues, we recently showed that CCR5 Nt binds gp120 through a conserved region specific for TYS moieties and suggested that this site may represent a target for inhibitors and probes of HIV-1 entry. As peptides containing sulfo-tyrosines are difficult to synthesize and handle due to limited stability of the sulfo-ester moiety, we have now incorporated TYS isosteres into CCR5 Nt analogs and assessed their binding to a complex of gp120-CD4 using saturation transfer difference (STD) NMR and surface plasmon resonance (SPR). STD enhancements for CCR5 Nt peptides containing tyrosine sulfonate (TYSN) in complex with gp120-CD4 were very similar to those observed for sulfated CCR5 Nt peptides indicating comparable modes of binding. STD enhancements for phosphotyrosine-containing CCR5 Nt analogs were greatly diminished consistent with earlier findings showing sulfo-tyrosine to be essential for CCR5 Nt binding to gp120. Tyrosine sulfonate-containing CCR5 peptides exhibited reduced water solubility, limiting their use in assay and probe development. To improve solubility, we designed, synthesized, and incorporated in CCR5 Nt peptide analogs an orthogonally functionalized azido tris(ethylenoxy) l-alanine (l-ate-Ala) residue. Through NMR and SPR experiments, we show a 19-residue TYSN-containing peptide to be a functional, hydrolytically stable CCR5 Nt isostere that was in turn used to develop both SPR-based and ELISA assays to screen for inhibitors of CCR5 binding to gp120-CD4.     

Optimization of the phenylurea moiety in a phosphoinositide 3-kinase (PI3K) inhibitor to improve water solubility and the PK profile by introducing a solubilizing group and ortho substituents

Phosphoinositide 3-kinase (PI3K) is a promising anti-cancer target, because various mutations and amplifications are observed in human tumors isolated from cancer patients. Our dihydropyrrolopyrimidine derivative with a phenylurea moiety showed strong PI3K enzyme inhibitory activity, but its pharmacokinetic property was poor because of lack of solubility. Herein, we report how we improved the solubility of our PI3K inhibitors by introducing a solubilizing group and ortho substituents to break molecular planarity.     

Antitumor agents 290. Design, synthesis, and biological evaluation of new LNCaP and PC-3 cytotoxic curcumin analogs conjugated with anti-androgens

In our continuing study of curcumin analogs as potential anti-prostate cancer drug candidates, 15 new curcumin analogs were designed, synthesized and evaluated for cytotoxicity against two human prostate cancer cell lines, androgen-dependent LNCaP and androgen-independent PC-3. Twelve analogs (5-12, 15, 16, 19, and 20) are conjugates of curcumin (1) or methyl curcumin (2) with a flutamide- or bicalutamide-like moiety. Two compounds (22 and 23) are C4-mono- and difluoro-substituted analogs of dimethyl curcumin (DMC, 21). Among the newly synthesized conjugates compound 15, a conjugate of 2 with a partial bicalutamide moiety, was more potent than bicalutamide alone and essentially equipotent with 1 and 2 against both prostate tumor cell lines with IC(50) values of 41.8 μM (for LNCaP) and 39.1 μM (for PC-3). A cell morphology study revealed that the cytotoxicity of curcumin analogs or curcumin-anti-androgen conjugates detected from both prostate cancer cell lines might be due to the suppression of pseudopodia formation. A molecular intrinsic fluorescence experiment showed that 1 accumulated mainly in the nuclei, while conjugate 6 was distributed in the cytosol. At the tested conditions, anti-androgens suppressed pseudopodia formation in PC-3 cells, but not in LNCaP cells. The evidence suggests that distinguishable target proteins are involved, resulting in the different outcomes toward pseudopodia suppression.     

Structural studies on the diglyceride-mediated activation of protein kinase C

Diglyceride analogs were studied with respect to their abilities to activate protein kinase C (Ca2+- and phospholipid-dependent protein kinase) in the presence of low calcium and phospholipid. Analogs which lacked either a free hydroxyl group at the 3 position or an ester moiety at the 1 position were without activity. It was concluded that the hydrophilic moieties of the active diglycerides are crucial for activity. However, diglyceride analogs containing additional hydrophilic moieties in one of the acyl side chains did not exhibit enhanced activity when compared to diglycerides containing two fatty acyl groups. Diglyceride analogs with a modified glycerol backbone were also studied. Homologous diglycerides with either one or two methylene groups between the 3-methylene group of the diglyceride and the hydroxyl group possessed markedly reduced activities when compared to the appropriate unmodified diglyceride. Isomers of these homologues which contained either a methyl group at the 1 position, or dimethyl groups incorporated at the 1 and 3 positions, were virtually without activity. Where studied, none of the diglyceride analogs prepared possessed antagonist activity. The results of these experiments are discussed with respect to the extreme specificity observed.     

Acetyl analogs of combretastatin A-4: synthesis and biological studies

The combretastatins have received significant attention because of their simple chemical structures, excellent antitumor efficacy and novel antivascular mechanisms of action. Herein, we report the synthesis of 20 novel acetyl analogs of CA-4 (1), synthesized from 3,4,5-trimethoxyphenylacetone that comprises the A ring of CA-4 with different aromatic aldehydes as the B ring. Molecular modeling studies indicate that these new compounds possess a 'twisted' conformation similar to CA-4. The new analogs effectively inhibit the growth of human and murine cancer cells. The most potent compounds 6k, 6s and 6t, have IC(50) values in the sub-μM range. Analog 6t has an IC(50) of 182 nM in MDA-MB-435 cells and has advantages over earlier analogs due to its enhanced water solubility (456 μM). This compound initiates microtubule depolymerization with an EC(50) value of 1.8 μM in A-10 cells. In a murine L1210 syngeneic tumor model 6t had antitumor activity and no apparent toxicity.     

A comprehensive study of Sansalvamide A derivatives: The structure–activity relationships of 78 derivatives in two pancreatic cancer cell lines

We report an extensive structure–activity relationship (SAR) of 78 compounds active against two pancreatic cancer cell lines. Our comprehensive evaluation of these compounds utilizes SAR that allow us to evaluate which features of potent compounds play a key role in their cytotoxicity. This is the first report of 19 new second-generation structures, where these new compounds were designed from the first generation of 59 compounds. These 78 structures were tested for their cytotoxicity and this is the first report of their activity against two pancreatic cancer cell lines. Our results show that out of 78 compounds, three compounds are worth pursuing as leads, as they show potency of ?55% in both cancer cell lines. These three compounds all have a common structural motif, two consecutive d-amino acids and an N-methyl moiety. Further, of these three compounds, two are second-generation structures, indicating that we can incorporate and utilize data from the first generation to design potency into the second generation. Finally, one analog is in the mid nanomolar range, and has the lowest IC₅₀ of any reported San A derivative. These analogs share no structural homology to current pancreatic cancer drugs, and are cytotoxic at levels on par with existing drugs treating other cancers. Thus, we have established Sansalvamide A as an excellent lead for killing multiple pancreatic cancer cell lines.     

Contribution of pi-electrons to the inhibition of S-adenosyl-L-methionine decarboxylase by aromatic tricyclic compounds

Eleven structural analogs with norharmane (9H-pyrido [3, 4-b] indole) as the basic structure were used to study structure-activity relations of adenosyl-methionine decarboxylase inhibition by this type of compounds. All analogs with an intact or 3,4-dihydrogenated pyrido ring inhibited the enzyme competitively with apparent Kis at the micromolar range, but complete hydrogenation of the pyrido moiety abolished the inhibition. This indicates that a suitable arrangement of pi-electrons is an essential structural feature in the process. Strongest inhibitors (Ki approximately 10-5M) were compounds methoxy or hydroxy groups in positions 6 or 7 of the indole moiety, suggesting that polarizing groups containing atoms with free electron pairs also are important in the interaction. The results offer a new viewpoint when action mechanisms of the known adenosylmethionine decarboxylase inhibitors are evaluated and they may prove useful when new inhibitors to the enzyme are designed.     

Synthesis and biological activities of novel 17-aminogeldanamycin derivatives

Geldanamycin interferes with the action of heat shock protein 90 (Hsp90) by binding to the N-terminal ATP binding site and inhibiting an essential ATPase activity. In a program directed toward finding potent, water soluble inhibitors of Hsp90, we prepared a library of over sixty 17-alkylamino-17-demethoxygeldanamycin analogs, and compared their affinity for Hsp90, ability to inhibit growth of SKBr3 mammalian cells, and in selected cases, water solubility. Over 20 analogs showed cell growth inhibition potencies similar to that of 17-allylamino-17-demethoxygeldanamycin (17-AAG), the front-runner geldanamycin analog that is currently in multiple clinical trials. Many of these analogs showed water solubility properties that were desirable for formulation. One of the most potent and water-soluble analogs in the series was 17-(2-dimethylaminoethyl)amino-17-demethoxygeldanamycin (17-DMAG), which was independently prepared by the NCI and will soon enter clinical trials. Importantly, the binding affinity of these analogs to the molecular target Hsp90 does not correlate well with their cytotoxicity in SKBr3 cells.     

Partial [alphaMe]Aun scan of [l-Leu11-OMe]-trichogin GA IV, a membrane active synthetic precursor of the natural lipopeptaibol.

We synthesized using solution-phase methods three analogs of [l-Leu11-OMe] trichogin GA IV, a membrane active synthetic precursor of the lipopeptaibol antibiotic in which the N-terminal n-octanoyl group and each of the three Aib residues in positions 1, 4 and 8 are replaced by an acetyl group and the lipophilic Calpha,alpha-disubstituted glycine l-(alphaMe)Aun, respectively [partial (alphaMe)Aun scan]. FT-IR absorption and CD analyses unequivocally show that the main three-dimensional structural features of [l-Leu11-OMe] trichogin GA IV are preserved in the analogs. Also, [l-Leu11-OMe] trichogin GA IV and the three Nalpha-acetylated l-(alphaMe)Aun analogs exhibit strictly comparable membrane-modifying properties. Taken together, these results strongly favor the conclusion that a shift of the long hydrocarbon moiety from the Nalpha-blocking group to the side-chain of the 1, 4 or 8 residue does not have any significant effect on the conformational properties or the membrane activity of [l-Leu11-OMe] trichogin GA IV and, by extension, of the natural lipopeptaibol.     

Assessment of the bioactive conformation of the farnesyltransferase protein binding recognition motif by computational methods.

Ras farnesyltransferase catalyzes the carboxyl-terminal farnesylation of Ras as well as other proteins involved in signal transduction processes. Previous studies demonstrated that its inhibition suppresses the activity of Ras transformed phenotypes in cultured cells, causing tumor regression in animal models. This observation led to the consideration of farnesyltransferase as a target for cancer therapy. In the present work we report the results of a computational study aimed at assessing the bioactive conformation of the peptide Cys-Val-Phe-Met, known to be the minimum peptide sequence that inhibits farnesyltransferase. For this purpose the conformational preferences of four analogs of the peptide were assessed by means of thorough searches of their respective conformational spaces, using a simulated annealing protocol as sampling technique. Specifically, two active analogs: Cys-Val-Tic-Met and Cys-Val-psi(CH2NH)Tic-Met and two inactive analogs: Cys-Val-Tic-psi(CH2NH)Met and Cys-Val-Aic-Met were selected for the present study. Low energy conformations of the four analogs were classified according to their structural motifs. The putative bioactive conformation of the minimum farnesyltransferase recognition motif was assessed by cross-comparison of the different classes of conformations obtained for the two active and the two inactive analogs. The putative bioactive conformation is characterized by two structural motifs: i) a C14 pseudo-ring stabilized by a hydrogen bond between the amino group of Cys1 and the carboxylate group of Met4 and a C11 pseudo-ring involving the residues Cys1 and Tic3. In addition, the thiol group of Cys1 side chain of the bioactive conformation points to the carboxylate moiety of Met4.     

Discovery of new aminopyrimidine-based phosphoinositide 3-kinase beta (PI3Kβ) inhibitors with selectivity over PI3Kα

Phosphatidylinositol-3-kinase beta (PI3Kβ) is an important therapeutic target in arterial thrombosis and special types of cancer. In this study, a new series of aminopyridine-based PI3Kβ selective inhibitors have been developed by the structure-based design strategy. When incorporated with the phenyl ring on sulfonamide moiety, aminopyrimidine analogs showed good potency on PI3Kβ and selectivity over PI3Kα. Intriguingly, replacement of phenyl group on sulfonamide with naphthyl group enhanced selectivity over PI3Kα while retaining submicromolar PI3Kβ potency. Molecular modeling suggests that increased PI3Kβ specificity is caused by the interaction with salt bridge (Lys782-Asp923) and Asp862 that creat a unique pocket in PI3Kβ. These results clearly provide useful insight in the design of new PI3Kβ inhibitors with high potency and selectivity.     

Design,synthesis and evaluation of γ-turn mimetics as LSD1-selective inhibitors

Lysine-specific demethylase 1 (LSD1) is an attractive molecular target for cancer therapy. We have previously reported potent LSD1-selective inhibitors (i.e., NCD18, NCD38, and their analogs) consisting of trans-2-phenylcyclopropylamine (PCPA) or trans-2-arylcyclopropylamine (ACPA) and a lysine moiety that could form a γ-turn structure in the active site of LSD1. Herein we report the design, synthesis and evaluation of γ-turn mimetic compounds for further improvement of LSD1 inhibitory activity and anticancer activity. Among a series of γ-turn mimetic compounds synthesized by a Mitsunobu-reaction-based amination strategy, we identified 1n as a potent and selective LSD1 inhibitor. Compound 1n induced cell cycle arrest and apoptosis through histone methylation in human lung cancer cells. The γ-turn mimetics approach should offer new insights into drug design for LSD1-selective inhibitors.     

New effective inhibitors of the Abelson kinase

The effects of substituents on the aryl ring were studied by the preparation and testing of several PD173955 analogs. Inserting a single carbon atom into the C–N bond in the aniline subunit (PDC) reduced the kinase inhibition by a factor of 200. Despite its decreased affinity for Abl compared with PD173955, PDC exhibits a Ki very similar to that reported for Imatinib. Increased water solubility is also gained by replacing the thiomethyl group with an amino or glycyl moiety. For both PD173955 and PDC, the analogs with amino groups in place of the methylthio group are 10 times more inhibitory than the parent molecules. Two molecules were identified with Kis about three orders of magnitude lower than reported for Imatinib.     

Synthesis of novel di- and tricationic carbapenems with potent anti-MRSA activity

A new series of 1β-methyl carbapenems possessing a 6,7-disubstituted imidazo[5,1-b]thiazol-2-yl group directly attached to the C-2 position of the carbapenem nucleus was prepared, and their activities against methicillin-resistant Staphylococcus aureus (MRSA) were evaluated. First, a benzyl moiety was introduced at the C-6 position of imidazo[5,1-b]thiazole attached to the carbapenem. These benzylated molecules showed potent anti-MRSA activity, but poor water solubility. In order to overcome this drawback, we designed and synthesized di- and tricationic carbapenems and finally discovered a novel carbapenem (15i), which exhibited excellent anti-MRSA activity and good water solubility.     

NMR studies of new arginine vasopressin analogs modified with α-2-indanylglycine enantiomers at position 2 bound to sodium dodecyl sulfate micelles

In this paper, we use NMR spectroscopy and molecular modeling to examine four new vasopressin analogs modified with α-2-indanylglycine (Igl) at position 2, [L-Igl²]AVP (I), [D-Igl²]AVP (II), [Mpa¹,L-Igl²]AVP (III) and [Mpa¹,D-Igl²]AVP (IV), embedded in a sodium dodecyl sulfate (SDS) micelle. All the analogs display antiuterotonic activity. In addition, the analogs with D-Igl reveal antipressor properties.Each analog exhibits the tendency to adopt β-turns at positions 2, 3 and/or 3, 4, which is characteristic of oxytocin-like peptides. Mutual arrangement of aromatic residues at positions 2 and 3 has been found to be crucial for binding antagonists with the OT and V_1a receptors. The orientation of the Gln⁴ side chain seems to be important for the V_1a receptor affinity. In each of the peptides studied, the Gln⁴ side chain is folded back over the ring moiety. However, it lies on the opposite face of the tocin moiety in analogs with L and D enantiomers of Igl.     

Enzymatic Glycosylation of Nonbenzoquinone Geldanamycin Analogs via Bacillus UDP-Glycosyltransferase

Geldanamycin (GM) is a naturally occurring anticancer agent isolated from several strains of Streptomyces hygroscopicus. However, its potential clinical utility is compromised by its severe toxicity and poor water solubility. For this reason, considerable efforts are under way to make new derivatives that have both good clinical efficacy and high water solubility. On the other hand, glycosylation is often a step that improves the water solubility and/or biological activity in many natural products of biosynthesis. Here, we report the facile production of glucose-conjugated nonbenzoquinone GM analogs using the Bacillus UDP-glycosyltransferase BL-C. Five aglycon substrates containing nonbenzoquinone aromatic rings were chosen to validate the in vitro glycosylation reaction. Putative glucoside compounds were determined through the presence of a product peak(s) and were also verified using LC/MS analyses. Further, the chemical structures of new glucoside compounds 6 and 7 were elucidated using spectroscopy data. These glucoside compounds showed a dramatic improvement in water solubility compared with that of the original aglycon, nonbenzoquinone GM.     

The 5-substituted piperazine as a novel secondary pharmacophore greatly improving the physical properties of urea-based inhibitors of soluble epoxide hydrolase

The inhibition of the mammalian soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of hypertention and inflammation. The problems of limited water solubility and high melting points commonly displayed by the active 1,3-disubstituted ureas prevent the further development of potent urea-based sEH inhibitors. Therefore, a new class of potent inhibitors of sEH were designed and synthesized by the introduction of a polar constrained piperazino group in the right side of adasmantyl urea to increase the water solubility. A facile and general synthesis was established to prepare a series of 1-adamantan-1-yl-3-(2-piperazin-2-yl-ethyl)-ureas (1a-d) with various 5-substitutions on the 2-piperazino ring, which will advance the SAR study by the efficient making of structurally diverse analogs. The effect of the 5-substitution on the activity and the water solubility was examined. The best potency was exhibited by the 5-benzyl-substituted-piperazine-containing urea with an IC50 value of 1.37 microM against human sEH and good water solubility (S=7.46 mg/mL) and low melting point, in which the 5-substituted piperazine serves as a favorable secondary pharmacophore and a water-solubility enhancing group. Our present work provides a promising new template for the design of orally available therapeutic agents for the disorders that can be addressed by changing the in vivo concentration of the chemical mediators that contain an epoxide.     

Design, synthesis and initial biological evaluation of a novel pladienolide analog scaffold

A novel and simplified synthetic scaffold based on pladienolide was designed using a consensus pharmacophore hypothesis. An initial target was synthesized and evaluated to examine the role of the 3-hydroxy group and the methyl groups present at positions 10, 16, 20, 22 in 1, on biological activity. We report the first totally synthetic analog of this macrolide that shows biological activity. Our novel synthetic strategy enables the rapid synthesis of other new analogs of pladienolide in order to develop selective anticancer lead compounds.     

Water‐Soluble Cationic Derivatives of Indirubin,the Active Anticancer Component from Indigo naturalis

To overcome the problem of poor aqueous solubility and bioavailability of indirubin‐3‐oximes, the compounds were modified by attaching a quaternary ammonium group at the oxime moiety. Exploring the prodrug concept, an oxime ester with acetyl‐l‐ carnitine was prepared, and the rate of its hydrolysis was investigated to assess its suitability for clinical administration. In addition, the cytotoxic potency of new stable oxime ethers with a choline moiety and their influence on the cell cycle were tested in human cancer cell lines.