Stereoselective reduction of C-2 substituted steroid C-3 ketones with lithium tris-(R,S-1,2-dimethylpropyl)-borohydride and sodium borohydride

The effect of C-2 substitution on the stereoselective reduction of steroid C-3 ketones with lithium tris-(R,S-1,2-dimethylpropyl)-borohydride and sodium borohydride was investigated. The C-2 mono- and di-substituted chloro and methyl derivatives were predominantly reduced to one of the epimeric alcohols. The 2 alpha-chloro and 2 alpha-methyl derivatives of 17 beta-acetoxy-5 alpha-androstan-3-one undergo stereoselective reduction with lithium tris-(R,S-1,2-dimethylpropyl)-borohydride to the axial (3 alpha) alcohol as observed in the unsubstituted compound, whereas sodium borohydride gives predominantly the equatorial (3 beta) alcohol. The 2 beta-chloro, 2 beta-methyl, 2,2-dichloro, and 2,2-dimethyl derivatives are reduced predominantly to the equatorial (3 beta) alcohol by both reagents.     

Simultaneous regioselective protection of phenyl 1-thioglucosides at the C-3 and C-6 or at the C-2 and C-6 hydroxy groups

Simultaneous regioselective 3,6- or 2,6-selective protection of 1-thio-beta- or alpha-D-glucopyranosides is described. The C-3 and C-6 hydroxy groups of the beta-thioglucoside were selectively protected with triisopropylsilyl or tert-butyldiphenylsilyl trifluoromethanesulfonate. The C-2 and C-6 hydroxy groups of the alpha-thioglucoside were selectively protected with tert-butyldiphenylsilyl trifluoromethanesulfonate.     

Synthesis of beta-D-mannosides from beta-D-glucosides via an intramolecular SN2 reaction at C-2.

The selective synthesis of beta-D-mannosides was achieved by first synthesizing beta-D-glucosides that carry a N-phenylcarbamoyl protecting group at O-3. These derivatives were transformed into the corresponding beta-D-mannosides by intramolecular nucleophilic substitution with inversion of configuration at C-2, the O-trifyl group being the leaving group. Subsequent intramolecular attack of the neighboring carbamoyl group resulted in the formation of the 2,3-carbonate of the desired beta-D-mannoside.     

The development of novel C-2, C-8, and N-9 trisubstituted purines as inhibitors of TNF-alpha production

A series of C-2, C-8, and N-9 trisubstituted purine based inhibitors of TNF-alpha production are described. The most potent analogs showed low nanomolar activity against LPS-induced TNF-alpha production in a THP-1 cell based assay. The SAR of the series was optimized with the aid of X-ray co-crystal structures of these inhibitors bound with mutated p38 (mp38).     

Investigation of the specificity of an alpha-L-arabinofuranosidase using C-2 and C-5 modified alpha-L-arabinofuranosides

The synthesis of three novel glycosyl donors presenting the same scaffold as alpha-L-arabinofuranose but modified at the C-2 or C-5 positions has been achieved. Furthermore, chemoenzymatic syntheses using the alpha-L-arabinofuranosidase AbfD3 and these unnatural furanosides were investigated. The use of the novel p-nitrophenyl furanoside donors revealed that AbfD3 can perform transglycosylation with the C-5 deoxygenated donor but not with the C-2 modified one. These results emphasize the vital role for OH-2 in AbfD3 substrate recognition.     

Synthesis and biological evaluation of C-3'NH/C-10 and C-2/C-10 modified paclitaxel analogues

Concurrent modifications on the C-3'NH/C-10, and C-2/C-10 positions on paclitaxel were carried out as a way of investigating possible synergistic effects. The biological activities of these analogues were evaluated in both a microtubule assembly assay and human ovarian cancer (A2780) and prostate cancer (PC3) cytotoxicity assay. In some cases the doubly modified analogues were more active than would have been predicted based on the activity of the singly modified analogues, indicating probable synergistic effects.     

Chain termination and inhibition of Saccharomyces cerevisiae poly(A) polymerase by C-8-modified ATP analogs

The nucleotide substrate specificity of yeast poly(A) polymerase (yPAP) toward various C-2- and C-8-modified ATP analogs was examined. 32P-Radiolabeled RNA oligonucleotide primers were incubated with yPAP in the absence of ATP to assay polyadenylation using unnatural ATP substrates. The C-2-modified ATP analogs 2-amino-ATP and 2-chloro (Cl)-ATP were excellent substrates for yPAP. 8-Amino-ATP, 8-azido-ATP, and 8-aza-ATP all produced chain termination of polyadenylation, and no primer extension was observed with the C-8-halogenated derivatives 8-Br-ATP and 8-Cl-ATP. The effects of modified ATP analogs on ATP-dependent poly(A) tail synthesis by yPAP were also examined. Whereas C-2 substitution (2-amino-ATP and 2-Cl-ATP) had little effect on poly(A) tail length, C-8 substitution produced moderate (8-amino-ATP, 8-azido-ATP, and 8-aza-ATP) to substantial (8-Br-ATP and 8-Cl-ATP) reduction in poly(A) tail length. To model the biochemical consequences of 8-Cl-Ado incorporation into RNA primers, a synthetic RNA primer containing a 3'-terminal 8-Cl-AMP residue was prepared. Polyadenylation of this modified RNA primer by yPAP in the presence of ATP was blocked completely. To probe potential mechanisms of inhibition, two-dimensional NMR spectroscopy experiments were used to examine the conformation of two C-8-modified AMP nucleotides, 8-Cl-AMP and 8-amino-AMP. C-8 substitution in adenosine analogs shifted the ribose sugar pucker equilibrium to favor the DNA-like C-2'-endo form over the C-3'-endo (RNA-like) conformation, which suggests a potential mechanism for polyadenylation inhibition and chain termination. Base-modified ATP analogs may exert their biological effects through polyadenylation inhibition and thus may provide useful tools for investigating polyadenylation biochemistry within cells.     

Structure-activity relationships of a series of benzothiophene-derived NPY Y1 antagonists: optimization of the C-2 side chain

A series of benzo[b]thiophene-derived NPY-1 receptor antagonists is described. Systematic modification of the C-2 substituent afforded a 1000-fold range in Y1 receptor affinity. Appropriate substitution at the ortho and para positions of the C-2 phenyl ether produced a synergistic effect on Y1 binding affinity, which led to the discovery of the most active ligands, 12t (K(i) = 15 nM), 12u (K(i) = 11 nM), and 12v (K(i) = 13 nM).     

Chitosan grafted with macrocyclic polyamines on C-2 and C-6 positions as nonviral gene vectors: preparation, characterization, and in vitro transfection studies

Chitosan grafted with macrocyclic polyamines (Cs-g-MCPA) on the C-2 or the C-6 position was synthesized by a simple method. Four copolymers prepared were characterized by (1)H NMR, (13)C NMR, Fourier transform infrared spectra (FTIR), X-ray diffraction (XRD), and gel permeation chromatography (GPC). Circular dichroism spectra (CD), fluorescence spectra and agarose gel electrophoresis assay showed that Cs-g-MCPA copolymers had good binding ability to DNA. By modification of the MCPAs, the copolymers showed low cytotoxicity and high transfection efficiency as new gene vectors. It was found that Cs-g-MCPA copolymers with different grafted positions showed different properties: copolymers grafted on the C-2 position showed higher cytotoxicity and higher transfection efficiency than those grafted on position C-6.     

Synthesis and bioactivity of C-2 and C-3 methyl ether derivatives of brassinolide

The following six novel methyl ether derivatives of brassinolide were prepared and their brassinosteroid activity was measured by means of the rice leaf lamina inclination bioassay: 2-O-methylbrassinolide, 3-O-methylbrassinolide, 2,22,23-tri-O-methylbrassinolide, 3,22,23-tri-O-methylbrassinolide, 2-O-methyl-25-methoxybrassinolide and 3-O-methyl-25-methoxybrassinolide. Brassinolide was used as a standard for comparison. All six compounds were also tested in the presence of 1000 ng of indole-3-acetic acid (IAA), an auxin that synergizes the effects of brassinosteroids. The 2-O-methyl- and 3-O-methylbrassinolide derivatives showed weak activity at high doses, which was enhanced by IAA, especially in the case of the 3-O-methyl derivative. Similarly, the 2,22,23-tri-O-methyl- and 3,22,23-tri-O-methyl derivatives displayed weak bioactivity on their own, but significantly stronger activity when applied with IAA. The 3-O-methyl and 3,22,23-tri-O-methyl analogues plus IAA were comparable in bioacivity to brassinolide alone, but were less active than brassinolide plus IAA. In each case, O-methylation at C-2 resulted in a greater loss of activity than O-methylation at C-3 under the same conditions. The relatively strong activity of 3,22,23-tri-O-methylbrassinolide in the presence of IAA is especially noteworthy as it indicates that free hydroxyl groups at C-3, C-22, and C-23 are not essential for bioactivity. Finally, 2-O-methyl- and 3-O-methyl-25-methoxybrassinolide were essentially inactive alone, and showed only a modest increase in bioactivity when coapplied with IAA.     

Synthesis and biological evaluation of cryptophycin analogs with substitution at C-6 (fragment C region)

Analogs of the antitumor agents cryptophycins 1 and 8 with dialkyl substitution at C-6 (fragment C) were synthesized and evaluated for in vitro cytotoxicity against human leukemia cells (CCRF-CEM). The activity of these analogs decreased as the size of the substituents at C-6 increased. The C-6 spirocylopropyl compound (2g) was highly potent in vitro and showed excellent antitumor activity in animal models.     

Preparation of various C-2 branched carbohydrates using intramolecular radical reactions

A new and efficient method for the facile synthesis of C-2 branched carbohydrates has been developed using an intramolecular radical cyclization fragmentation reaction. The desired C-2 branched glucopyranosides were isolated in 40-84% yield. Additionally, an unexpected furanoside was obtained from a tributyltin iodide-promoted rearrangement of the radical intermediate. The C-2 formyl glycal was also isolated in good yield using tris(trimethylsilyl)silane (TTMSS) as the reducing agent. This method was extended to synthesize a beta C-2 branched glucopyranoside, a C-2 branched galactoside and a C-2 cyano glucopyranoside.     

Further characterization of the 2-deoxyecdysone C-2 hydroxylase from Locusta migratoria

Additional data are provided on the enzyme 2-deoxyecdysone C-2 hydroxylase which has been shown in a previous study (Kappler et al., 1986) to be a mitochondrial hydroxylase with some classical characteristics of a cytochrome P-450 monooxygenase but which appeared to be insensitive to CO. Using ¹⁸O₂, we have now demonstrated that molecular oxygen is directly incorporated into ecdysone during the process of C-2 hydroxylation. Neither cumene hydroperoxide nor linoleyl hydroperoxide could support C-2 hydroxylation. When the reaction was sustained by α-ketoglutarate, addition of cofactors like Fe²⁺, ascorbate and catalase caused only a slight increase of the enzymatic activity whereas the α-ketoglutarate-dependent hydroxylation was largely decreased in the presence of malonate; these data eliminate the possible existence of a dioxygenase mechanism for C-2 hydroxylation.The paper also provides inhibition kinetics which indicate that 2-deoxy-20-hydroxyecdysone, 2,22-bisdeoxyecdysone and 2,22,25-trideoxyecdysone are competitive inhibitors of the C-2 hydroxylase whereas the 3-epi isomer of 2-deoxyecdysone is a non-competitive inhibitor.     

Synthesis of some derivatives of C-(1-deoxy-1-N-substituted-D-glucopyranosyl)formic acid (D-gluco-hept-2-ulopyranosonic acid) as potential inhibitors of glycogen phosphorylase

Per-O-benzoylated derivatives (amide, methyl ester and glycinamide) of C-(1-azido-1-deoxy-alpha-D-glucopyranosyl)formic acid obtained by azide substitution in the corresponding C-(1-bromo-1-deoxy-beta-D-glucopyranosyl)formic acid derivatives were debenzoylated by the Zemplén-protocol. Per-O-benzoylated C-(1-azido-1-deoxy-alpha-D-glucopyranosyl)formamide was dehydrated by oxalyl chloride-DMF to give the corresponding nitrile, while from its reduction mixture obtained by Raney-nickel or sodium hydrogentelluride C-(1-amino-1-deoxy-beta-D-glucopyranosyl)formamide could be isolated. Acetylation of this amino-amide by Ac2O/Py and subsequent debenzoylation gave C-(1-acetamido-1-deoxy-beta-D-glucopyranosyl)formamide. Applying the same conditions to the crude reduction mixture allowed the alpha-anomer to be isolated as a minor component. An alternative pathway to produce the above beta-anomer appeared in the reaction of C-(1-bromo-1-deoxy-beta-D-glucopyranosyl)formamide with CH3CN in the presence of Ag2CO3 to yield 1-acetamido-2,3,4,6,-tetra-O-benzoyl-1-deoxy-beta-D-glucopyranosyl cyanide, which was hydrated, in the presence of TiCl4, to the formamide. Some of the new compounds were shown to be weak inhibitors of muscle glycogen phosphorylase b.     

Specificity of acceptor binding to Leuconostoc mesenteroides B-512F dextransucrase: binding and acceptor-product structure of alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 by inversion of the hydroxyl and by replacement of the hydroxyl with hydrogen

The specificity of acceptor binding to the active site of dextransucrase was studied by using alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 positions by (a) inversion of the hydroxyl group and (b) replacement of the hydroxyl group with hydrogen. 2-Deoxy-alpha-methyl-D-glucopyranoside was synthesized from 2-deoxyglucose; 3- and 4-deoxy-alpha-methyl-D-glucopyranosides were synthesized from alpha-methyl-D-glucopyranoside; and alpha-methyl-D-allopyranoside was synthesized from D-glucose. The analogs were incubated with [14C]sucrose and dextransucrase, and the products were separated by thin-layer chromatography and quantitated by liquid scintillation spectrometry. Structures of the acceptor products were determined by methylation analyses and optical rotation. The relative effectiveness of the acceptor analogs in decreasing order were 2-deoxy, 2-inverted, 3-deoxy, 3-inverted, 4-inverted, and 4-deoxy. The enzyme transfers D-glucopyranose to the C-6 hydroxyl of analogs modified at C-2 and C-3, to the C-4 hydroxyl of 4-inverted, and to the C-3 hydroxyl of 4-deoxy analogs of alpha-methyl-D-glucopyranoside. The data indicate that the hydroxyl group at C-2 is not as important for acceptor binding as the hydroxyl groups at C-3 and C-4. The hydroxyl group at C-4 is particularly important as it determines the binding orientation of the alpha-methyl-D-glucopyranoside ring.     

Preparation of methyl 2,3-di-O-mesyl-4,6-thioanhydro-alpha-D-galactopyranoside and methyl 2-O-mesyl-4,6-thioanhydro-alpha-D-gulopyranoside

Two 2-oxa-7-thiabicyclo[4.2.0]octane derivatives, 4 and 10, with the D-galacto and D-gulo configuration, respectively, were obtained from methyl alpha-D-glucopyranoside. The thietane cyclization involved a thio-Mitsunobu reaction resulting in a 6-thioacetate, which underwent selective base-catalyzed intramolecular nucleophilic substitution at a C-4 mesylate. The structures of 4 and 10 were elucidated by X-ray diffraction analysis.     

Structure-based discovery of C-2 substituted imidazo-pyrrolopyridine JAK1 inhibitors with improved selectivity over JAK2

Herein we describe our successful efforts in obtaining C-2 substituted imidazo-pyrrolopyridines with improved JAK1 selectivity relative to JAK2 by targeting an amino acid residue that differs between the two isoforms (JAK1: E966; JAK2: D939). Efforts to improve cellular potency by reducing the polarity of the inhibitors are also detailed. The X-ray crystal structure of a representative inhibitor in complex with the JAK1 enzyme is also disclosed.     

Anti-MRSA cephems. Part 2: C-7 cinnamic acid derivatives

Forty-five novel cephalosporin derivatives with activity against methicillin-resistant Staphylococcus aureus (MRSA) are described. The compounds contain novel cinnamic acid moieties at C-7 that were synthesized using a key Heck reaction followed by nucleophilic aromatic substitution reactions. The most active compound (41) displayed an MIC(90) against MRSA of 1.0 microg/mL, and a PD(50) of 0.8 mg/kg. Compound 14 was found to be very safe in a mouse model of acute toxicity.     

TEMPO-mediated selective oxidation of substituted polysaccharides--an efficient approach for the determination of the degree of substitution at C-6

2,2,6,6-Tetramethyl-1-piperidinyloxy radical (TEMPO)-mediated oxidations of substituted polysaccharides were studied at pH 10.2 and at a temperature of 0 °C with NaOCl as the oxidant. The reaction is highly selective, and it was shown that the oxidation can proceed to a yield of nearly 100%. The oxidation process was investigated for several substituted polysaccharides, especially for a series of hydroxypropyl guar gums with different molar degrees of substitution. It was shown that this oxidation can be used for the determination of the degree of substitution at C-6 of the polysaccharide by comparing the difference in oxidation yield between substituted and natural polysaccharides. Studies on several hydroxypropyl guar gums showed that the degrees of substitution at C-6—for MS of 0.08, 0.34, 0.62, and 1.08—are 0.06, 0.24, 0.40, and 0.44, respectively. The results were extended to other polysaccharides such as carboxymethyl cellulose, cationic guar gum, carboxymethyl pullulan, and methyl cellulose. It can be concluded that the TEMPO-mediated oxidation is a useful method for the determination of the DS at the substituted C-6 position for different kinds of modified polysaccharides.     

3-Methylene-substituted androgens as novel aromatization inhibitors. Evidence of a requirement for C-3 oxygen in C-19 hydroxylations

Substitution of a methylene group for the C-3 oxygen in androstenedione, testosterone, and the corresponding 19-hydroxy and 19-oxo derivatives results in a new category of inhibitors of estrogen biosynthesis by human placental microsomes. The inhibition is of the competitive type with the most effective inhibitors being the 17-ketonic compounds, 3-methyleneandrost-4-en-17-one, 19-hydroxy-3-methyleneandrost-4-en-17-one, and 3-methylene-19-oxoandrost-4-en-17-one with apparent Ki values of 4.7, 13, and 24 nM, respectively. The 3-methylene derivatives of androstenedione and 19-hydroxyandrostenedione were effective substrates for the placental microsomal 17 beta-hydroxy-steroid oxidoreductase but were only marginally hydroxylated at the C-19 position to the respective 19-hydroxy and 19-oxo derivatives. The 3-methylene analogs are thus competitive inhibitors of aromatization but are not substrates for this enzyme complex. Time-dependent inhibition of aromatization by 10 beta-difluoromethylestr-4-ene-3,17-dione and 10 beta-(2-propynyl)estr-4-ene,3,17-dione was abolished by substitution of a methylene function for the C-3 oxygen, suggesting that the presence of an oxygen at C-3 is required for an oxidative transformation at C-19, an initial step in aromatization. The essential role of the C-19 hydroxylation in aromatization is supported by the observation that the 3-methylene derivatives of 19-hydroxy- and 19-oxoandrostenedione showed time-dependent inhibition, but the corresponding 19-methyl compound did not. The 3-methylene androgens are potent inhibitors of placental aromatization but are themselves only marginal substrates for the enzyme. Their high affinity for and inertness to the placental aromatase complex makes them valuable probes of the aromatization process.