Functional studies of rat hydroxymethylbilane synthase

The structurally related tetrapyrrolic pigments are a group of natural products that participate in many of the fundamental biosynthetic and catabolic processes of living organisms. Hydroxymethylbilane synthase catalyzes a rate-limiting step for the biosyntheses of tetrapyrrolic natural products. We carried out extensive studies of rat hydroxymethylbilane synthase in the present investigation. The enzymatic reaction rate of the holoenzyme was found to be lower than those of the enzyme-intermediate complexes, which corrected the previous theoretical analysis result. Several mutants were constructed, purified and characterized. D44 was found to play an important role in the disassembly of the enzyme-intermediate complexes. E63 and H78 were important for maintaining the activity of the enzyme at high temperature. Four substrate analogs with variation of porphobilinogen side-chain were synthesized and incubated with the enzyme. Three analogs were found to be weak substrates of the enzyme. All four analogs can be used for the preparation of uroporphyrin I analogs.     

Biological evaluation of 3'-O-alkylated analogs of salacinol, the role of hydrophobic alkyl group at 3' position in the side chain on the α-glucosidase inhibitory activity

Four analogs with 3′-O-alkyl groups (9a: CH₃, 9b: C₂H₅, 9c: C₁₃H₂₇ or 9d: CH₂Ph) instead of the 3′-O-sulfate anion in salacinol (1), a naturally occurring potent α-glucosidase inhibitor, were synthesized by the coupling reaction of 1,4-dideoxy-1,4-epithio-d-arabinitols (18a and 18b) with appropriate epoxides (10a-10d). These analogs showed equal or considerably higher inhibitory activity against rat small intestinal α-glucosidases than the original sulfate (1), and one of them (9d) was found more potent than currently used α-glucosidase inhibitors as antidiabetics. Thus, introduction of a hydrophobic moiety at the C3′ position of this new class of inhibitor was found beneficial for onset of stronger inhibition against these enzymes.     

Synthesis and evaluation of isatin analogs as caspase-3 inhibitors: introduction of a hydrophilic group increases potency in a whole cell assay

A series of isatin analogs containing a hydrophilic group, including a pyridine ring, ethylene glycol group, and a triazole ring, have been synthesized, and their inhibition potency for caspase-3 was measured both in vitro (i.e., recombinant enzyme) and in whole cells (HeLa cells). The analogs having a hydrophilic group, including 12, 13, 16, 38, and 40, have dramatically increased activity in vitro and in HeLa cells compared to the corresponding unsubstituted N-phenyl isatin analogs.     

Synthetic analogs of daidzein, having more potent osteoblast stimulating effect

A series of didzein derivatives were synthesized and assessed for stimulation of osteoblast differentiation using primary cultures of rat calvarial osteoblasts. Data suggested that three synthetic analogs, 1c, 3a and 3c were several folds more potent than daidzein in stimulating differentiation and mineralization of osteoblasts. Further, these three compounds did not show any estrogen agonistic activity, however had mild estrogen antagonistic effect. Out of the three compounds, 3c was found to maximally increase the mineralization of bone marrow osteoprogenitor cells. Compound 3c also robustly increased the mRNA levels of osteogenic genes including bone morphogenetic protein-2 and osteocalcin in osteoblasts. Unlike daidzein, 3c did not inhibit osteoclastogenesis. Collectively, we demonstrate osteogenic activity of daidzein analogs at significantly lower concentrations than daidzein.     

Antidotes to anthrax lethal factor intoxication. Part 2: structural modifications leading to improved in vivo efficacy

New anthrax lethal factor inhibitors (LFIs) were designed based upon previously identified potent inhibitors 1a and 2. Combining the new core structures with modifications to the C2-side chain yielded analogs with improved efficacy in the rat lethal toxin model.     

Synthesis of 2-substituted 17β-hydroxy/17-methylene estratrienes and their in vitro cytotoxicity in human cancer cell cultures

Synthesis of various types of 2-(alkylaminomethyl) and 2-(aroyl) 17β-estradiol analogs are reported. The synthesis of similar types of 2-substituted 17-methylene estratriene analogs was also achieved. Synthesis of chalcone derivatives of 17β-estradiol and 17-methylene estratriene were also realized. All these 2-substituted estratrienes were tested for their antiproliferative activity by using four different cell lines from colon, lung, glioma and breast cancers. Among the various 2-substituted estratrienes, the compounds 10d, 14a-h and 17e were found to have in vitro antiproliferative activity comparable to that of parent analogs 1-4. Comparison of the SAR pattern of these 2-susbtituted estratriene derivatives confirmed that relatively, 17-methylene estratrienes are more active than that of 17β-estradiol analogs.     

Synthesis, structure-activity relationship and biological evaluation of novel N-substituted matrinic acid derivatives as host heat-stress cognate 70 (Hsc70) down-regulators

Oxymatrine (1) is a natural anti-hepatitis B virus (HBV) drug that down-regulates host heat-stress cognate 70 (Hsc70) expression through a mechanism different from that of nucleosides. Taking Hsc70 as a target against HBV, 26 novel N-substituted matrinic acid analogs were designed, synthesized and evaluated for their regulation of Hsc70 mRNA expression with 1 as the lead. The SAR analysis revealed that (i) the carboxyl group at the 11-position was required for activity; (ii) introducing of a substituent on the nitrogen atom at the 12-position of 3, especially substituted benzyl, might significantly improve the activity. Among these analogs, compound 9p possessing N-p-methoxylbenzyl afforded an increased anti-HBV effect in comparison with 1. We consider 9p a promising anti-HBV candidate.     

Synthesis and evaluation of chromenyl barbiturates and thiobarbiturates as potential antitubercular agents

A novel series of barbiturate and thiobarbiturate analogs of 2-benzoyl-3-methyl-5-oxo-5H-furo[3,2-g]chromene-6-carbaldehydes (3a-g and 4a-d, respectively) and 6-methyl-4,8-dioxo-4,8-dihydropyrano[3,2-g]chromenes (7a-c), were synthesized and evaluated for their antitubercular activities against Mycobacterium tuberculosis H37RV, and cytotoxicity (CC₅₀) in the VERO cell MABA assay. The results indicate that the furanochromene series of compounds (3a-g and 4a-d) showed only weak to moderate antitubercular activity. However, the pyranochromene analog 7b showed good antitubercular activity (IC₉₀: 5.9 μg/mL) and cytotoxicity (CC₅₀: 14.27 μg/mL). The antitubercular activity of 7b was superior to the antituberculosis drug, pyrazinamide (PZA; IC₉₀: >20 μg/mL). Analog 7b was considered to be a lead compound for subsequent structural optimization.     

Side chain SAR of bicyclic β-lactamase inhibitors (BLIs). 2. N-Alkylated and open chain analogs of MK-8712

The bridged monobactam β-lactamase inhibitor MK-8712 (1) effectively inhibits class C β-lactamases. Side chain N-alkylated and ring-opened analogs of 1 were prepared and evaluated for combination with imipenem to overcome class C β-lactamase mediated resistance. Although some analogs were more potent inhibitors of AmpC, none exhibited better synergy with imipenem than 1.     

Novel EGFR inhibitors prepared by combination of dithiocarbamic acid esters and 4-anilinoquinazolines

On the basis of combination strategy, a novel series of EGFR inhibitors were designed and synthesized by combination of dithiocarbamic acid esters and 4-anilinoquinazolines. The effect of the synthesized compounds on cell proliferation was evaluated by MTT assay in three human cancer cell lines: MDA-MB-468, SK-BR-3 and HCT-116. Two compounds (11d and 11f) were found more potent against all three cell lines and five compounds (11a, 11d-11g) were found more potent against both MDA-MB-468 and SK-BR-3 than Lapatinib. SAR studies revealed that the substituents on C6 and C7 positions of quinazoline, the amine component of dithiocarbamate moiety and the linker greatly affected the activity. This work provides a promising new strategy for the preparation of potent tyrosine kinase inhibitors.     

Antitumor agents 287. Substituted 4-amino-2H-pyran-2-one (APO) analogs reveal a new scaffold from neo-tanshinlactone with in vitro anticancer activity

4-Amino-2H-benzo[h]chromen-2-one (ABO) and 4-amino-7,8,9,10-tetrahydro-2H-benzo[h]chromen-2-one (ATBO) analogs were found to be significant in vitro anticancer agents in our previous research. Our continuing study has now discovered a new simplified (monocyclic rather than tricyclic) class of cytotoxic agents, 4-amino-2H-pyran-2-one (APO) analogs. By incorporating various substituents on the pyranone ring, we have established preliminary structure-activity relationships (SAR). Analogs 19, 20, 23, and 26-30 displayed significant tumor cell growth inhibitory activity in vitro. The most active compound 27 exhibited ED₅₀ values of 0.059-0.090 μM.     

Structural requirements of (E)-6-benzylidene-4a-methyl-4,4a,5,6,7,8-hexahydronaphthalen-2(3H)-one derivatives as novel melanogenesis inhibitors

Chalcone type compound 1a ((E)-6′-benzylidene-4a′-methyl-4′,4a′,7′,8′-tetrahydro-3′H-spiro[[1,3]dithiolane-2,2′-naphthalen]-5′(6′H)-one) was discovered as an potent inhibitor in melanogenesis. To define its structure-activity relationship, a series of analogs 1b-n, dithiolane truncated 2a-b and ring A removed 3a-e were prepared and evaluated. The electron donating substitution on the phenyl ring (ring C) rather than an electron withdrawing group and dithiolane motif of 1 are needed for the activity enhancement. The scaffold containing both rings A and B associated with α,β-unsaturated system connected to phenyl of 1 was essential for antimelanogenesis.     

Spirodiketopiperazine-based CCR5 antagonist: discovery of an antiretroviral drug candidate

Following the discovery that hydroxylated derivative 3 (Fig. 1) was one of the oxidative metabolites of the original lead 1, it was found that hydroxylated compound 4 possesses higher in vitro anti-HIV potency than the corresponding non-hydroxylated compound 2. Structural hybridation of 4 with the orally available analog 5 resulted in another orally-available spirodiketopiperazine CCR5 antagonist 6a that possesses more favorable pharmaceutical profile for use as a drug candidate.     

Synthesis, transport and antiviral activity of Ala-Ser and Val-Ser prodrugs of cidofovir

We report the synthesis and biological evaluation of Ala-(Val-)l-Ser-CO₂R prodrugs of 1, where a dipeptide promoiety is conjugated to the P(OH)₂ group of cidofovir (1) via esterification by the Ser side chain hydroxyl group and an ethyl group (4 and 5) or alone (6 and 7). In a murine model, oral administration of 4 or 5 did not significantly increase total cidofovir species in the plasma compared to 1 or 2, but 7 resulted in a 15-fold increase in a rat model and had an in vitro EC₅₀ value against human cytomegalovirus comparable to 1. Neither 6 nor 7 exhibited toxicity up to 100 μM in KB or HFF cells.     

Determination of the hydroxycinnamate profile of 12 members of the Asteraceae family

The hydroxycinnamates of the leaves of 12 plants of the Astreraceae family, Achillea millefolium, Arnica montana, Artemesia dracunculus, Cichorium intybus, Cnicus benedictus, Cynara scolymus, Echinops humilis, Inula helenium, Lactuca sativa, Petasites hybridus, Solidago virgaurea, and Tanacetum parthenium were investigated qualitatively by LC-MSⁿ. Thirty-nine chlorogenic acids were detected and all characterized to regioisomeric level on the basis of their fragmentation pattern in the tandem MS spectra, most of them for the first time from these sources with two of them previously not reported in nature. Both chlorogenic acids based on trans and cis-cinnamic acid substituents were identified. Assignment to the level of individual regioisomers was possible for seven caffeoylquinic acids (1-7), 11 dicaffeoylquinic acids (17-27), six feruloylquinic acids (9-14), two p-coumaroylquinic acids (15-16), two caffeoyl-feruloylquinic acids (28 and 29), four caffeoyl-p-coumaroylquinic acids (30-33), three dicaffeoyl-succinoylquinic acids (34-36), two dicaffeoyl-methoxyoxaloylquinic acids (37 and 38), and one tricaffeoylquinic acid (39). Furthermore, one caffeoylshikimic acid (40), one caffeoyltartaric acid (41), three dicaffeoyltartaric acids (42-44), and three caffeoyl-feruloyltartaric acids (45-47) were detected and shown to possess characteristic tandem MS spectra and were tentatively assigned on the basis of their retention time and previously developed hierarchical keys.     

5α,8α-Epidioxysterols from the gorgonian Eunicella cavolini and the ascidian Trididemnum inarmatum: Isolation and evaluation of their antiproliferative activity

Three new (1, 4, 9) and nine previously reported (2, 3, 5-8, 10-12) 5α,8α-epidioxysterols were isolated from the organic extracts of the gorgonian Eunicella cavolini and the ascidian Trididemnum inarmatum. The structures and relative configurations of 1-12 were established on the basis of detailed NMR spectroscopic analyses and comparison with the literature. The growth inhibitory effects of 1-12 were evaluated against MCF-7 human breast cancer cells. Compound 1, bearing a cyclopropyl moiety in the side chain, exhibited the highest antiproliferative activity.     

Design, synthesis, and evaluation of bromo-retrochalcone derivatives as protein tyrosine phosphatase 1B inhibitors

A series of bromo-retrochalcones was designed, synthesized and evaluated as PTP1B inhibitors based on licochalcone A and E. Compounds 6, 12, 13, 14, 25, 36, 37, 39, and 41 showed potent inhibitory effects against PTP1B, and compound 37, the most potent among the series, had an IC₅₀ value of 1.9 μM, about two-fold better than that of the positive control, ursolic acid.     

Synthesis and olfactory activity of unnatural, sulfated 5β-bile acid derivatives in the sea lamprey (Petromyzon marinus)

A variety of unnatural bile acid derivatives (9a-9f) was synthesized and used to examine the specificity with which the sea lamprey (Petromyzon marinus) olfactory system detects these compounds. These compounds are analogs of petromyzonol sulfate (PS, 1), a component of the sea lamprey migratory pheromone. Both the stereochemical configuration at C5 (i.e., 5α vs. 5β) and the extent and sites of oxygenation (hydroxylation or ketonization) of the bile acid derived steroid skeleton were evaluated by screening the compounds for olfactory activity using electro-olfactogram recording. 5β-Petromyzonol sulfate (9a) elicited a considerable olfactory response at sub-nanomolar concentration. In addition, less oxygenated systems (i.e., 9b-9e) elicited olfactory responses, albeit with less potency. The sea lamprey sex pheromone mimic 9f (5β-3-ketopetromyzonol sulfate) was also examined and found to produce a much lower olfactory response. Mixture studies conducted with 9a and PS (1) suggest that stimulation is occurring via similar modes of activation, demonstrating a relative lack of specificity for recognition of the allo-configuration (i.e., 5α) in sea lamprey olfaction. This attribute could facilitate design of pheromone analogs to control this invasive species.     

Investigation on the coordination modes: Syntheses, characterization and crystal structures of diorganotin (IV) dichloride with 4(5)-imidazoledithiocarboxylic acid

A series of diorganotin (IV) complexes of the types of R₂SnCl(SSCC₃H₃N₂) (R = CH₃1, ⁿBu 2, C₆H₅3 and C₆H₅CH₂4), R₂Sn(SSCC₃H₃N₂)₂ (R = CH₃5, ⁿBu 6, C₆H₅7 and C₆H₅CH₂8) and R₂Sn(SSCC₃H₂N₂) (R = CH₃9, ⁿBu 10, C₆H₅11 and C₆H₅CH₂12) have been obtained by reactions of 4(5)-imidazoledithiocarboxylic acid with diorganotin (IV) dichlorides in the presence of sodium ethoxide. All complexes are characterized by elemental, IR, ¹H, ¹³C and ¹¹⁹Sn NMR spectra analyses. Also, the complexes 1, 7 and 9 are characterized by X-ray crystallography diffraction analyses, which reveal that the complex 1 is monomeric structure with five-coordinate tin (IV) atom, the complex 7 is monomeric structure with six-coordinate tin (IV) atom and the complex 9 is one-dimensional chain with five-coordinate tin (IV) atom.