In vivo effective dibenzo[b,d]furan-1-yl-thiazoles as novel PDE-4 inhibitors.

Herein we report the synthesis, PDE-4B and TNF-α inhibitory activities of a few dibenzo[b,d]furan-1-yl-thiazole derivatives. The hydroxycyclohexanol amide derivatives 14, 18, 24, 29, 31 and 33 exhibited promising in vitro PDE-4B and TNF-α inhibitory activities. Compound 24 showed good systemic availability in preclinical animal models and was also found to be non-toxic (exploratory mutagenicity test). Further it exhibited promising results in in vivo asthma/COPD and Uveitis models.

Molecular details of quinolone-DNA interactions: solution structure of an unusually stable DNA duplex with covalently linked nalidixic acid residues and non-covalent complexes derived from it.

Quinolones are antibacterial drugs that are thought to bind preferentially to disturbed regions of DNA. They do not fall into the classical categories of intercalators, groove binders or electrostatic binders to the backbone. We solved the 3D structure of the DNA duplex (ACGCGU-NA)2, where NA denotes a nalidixic acid residue covalently linked to the 2'-position of 2'-amino-2'-deoxyuridine, by NMR and restrained torsion angle molecular dynamics (MD). In the complex, the quinolones stack on G:C base pairs of the core tetramer and disrupt the terminal A:U base pair. The displaced dA residues can stack on the quinolones, while the uracil rings bind in the minor groove. The duplex-bridging interactions of the drugs and the contacts of the displaced nucleotides explain the high UV-melting temperature for d(ACGCGU-NA)2 of up to 53 degrees C. Further, non-covalently linked complexes between quinolones and DNA of the sequence ACGCGT can be generated via MD using constraints obtained for d(ACGCGU-NA)2. This is demonstrated for unconjugated nalidixic acid and its 6-fluoro derivative. The well-ordered and tightly packed structures thus obtained are compatible with a published model for the quinolone-DNA complex in the active site of gyrases.

Clamping down on weak terminal base pairs: oligonucleotides with molecular caps as fidelity-enhancing elements at the 5'- and 3'-terminal residues.

The base-pairing fidelity of oligonucleotides depends on the identity of the nucleobases involved and the position of matched or mismatched base pairs in the duplex. Nucleobases forming weak base pairs, as well as a terminal position favor mispairing. We have searched for 5'-appended acylamido caps that enhance the stability and base-pairing fidelity of oligonucleotides with a 5'-terminal 2'-deoxyadenosine residue using combinatorial synthesis and MALDI-monitored nuclease selections. This provided the residue of 4-(pyren-1-yl)butyric acid as a lead. Lead optimization gave (S)-N-(pyren-1-ylmethyl)pyrrolidine-3-phosphate as a cap that increases duplex stability and base-pairing fidelity. For the duplex of 5'-AGGTTGAC-3' with its fully complementary target, this cap gives an increase in the UV melting point T(m) of +10.9 degrees C. The T(m) is 6.3-8.3 degrees C lower when a mismatched nucleobase faces the 5'-terminal dA residue. The optimized cap can be introduced via automated DNA synthesis. It was combined with an anthraquinone carboxylic acid residue as a cap for the 3'-terminal residue. A doubly capped dodecamer thus prepared gives a melting point decrease for double-terminal mismatches that is 5.7-5.9 degrees C greater than that for the unmodified control duplex.

5'-Tethered stilbene derivatives as fidelity- and affinity-enhancing modulators of DNA duplex stability.

A series of 5'-linked stilbene-DNA conjugates with different substituents in the distal aromatic ring of the stilbene was prepared, and the effect of the modifications on duplex stability was determined via UV-melting curves. A trimethoxystilbene derivative as a 5'-substituent increases duplex melting points by up to 12.2 degrees C per modification. With this alkoxystilbene substituent, terminal mismatches in DNA duplexes lower the melting point by up to 23.4 degrees C over the perfectly matched control, whereas terminal mismatches in unmodified DNA cause melting point depressions of no more than 6.1 degrees C. An aminomethylstilbene substituent linked to an oligopyrrolamide minor groove binder increases the melting point of an all-A/T decamer by up to 32.7 degrees C, thus shifting the melting point into a range typical for duplexes with statistical G/C-content. An affinity- and selectivity-enhancing effect was also observed when the trimethoxystilbene cap was employed on a small DNA microarray. The phosphoramidite of the trimethoxystilbene can be readily employed in automatic DNA synthesis, facilitating the generation of DNA chips with improved fidelity.

CpG oligonucleotides with modified termini and nicked dumbbell structure show enhanced immunostimulatory activity.

A series of 21 phosphodiester oligodeoxyribonucleotides containing the core sequence 5'-GACGTT-3' or related control sequences were prepared and tested for their immunostimulatory effect on murine macrophages. The range of structural modifications tested included substituents at 3'- or 5'-termini, N3-methylation of thymidine residues, and hexaethylene glycol linkers favoring nicked or cyclic dumbbell duplexes. Lipophilic and cationic substituents at the termini failed to increase the release of TNF-alpha and nitric oxide, but two new types of modification were found that enhance the stimulation of RAW264.7 macrophages. One is the substitution of the 5'-terminal hydroxyl group with an amino group, and the other is the introduction of linkers favoring nicked duplexes. Even for sequences without linkers, UV-melting analysis and two-dimensional NMR showed that the core sequence 5'-GACGTT-3' readily forms a duplex. The cyclic derivative of the most active nicked dumbbell sequence is inactive, however. Together these results suggest a recognition of both the 5'-terminus and the core of the CpG oligonucleotides by the putative receptor(s) and provide an entry into a class of modified oligonucleotides whose activity rivals that of phosphorothioates, but consists of synthetic compounds that are single stereoisomers.

Synthesis of oligonucleotides with a 2'-cap at the 3'-terminus via reversed phosphoramidites.

[reaction: see text] A method is presented for the synthesis of single compounds or small combinatorial libraries of oligonucleotides with 2'-acylamido-2'-deoxyuridine residues at the 3'-terminus. Selection experiments identified the residue of anthraquinone-2-carboxylic acid as a "molecular cap" that increases the UV melting point of the duplex (5'-ACGCGU-3')(2) by up to 28 degrees C compared to the unmodified control duplex.