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Detailed process safety investigation of an old process revealed hidden, previously unnoticed danger. The synthesis of morpholine-adduct 2 was performed under reaction conditions very close to trig...

5-Bromo-2-nitropyridine was prepared from the corresponding amine via hydrogen peroxide oxidation in large scale production. This transformation initially showed low conversion, high impurity content and lack of reproducibility in lab trials. Parallel to process development, safety studies were conducted to investigate the stability of oxidant mixture, its composition and the oxidation reaction itself by reaction and adiabatic calorimetry. The resulting robust reaction conditions and appropriate safety boundaries allowed to develop a reproducible, safe protocol for the implementation of this chemistry on large scale, obtaining consistent results throughout the campaign.

Suberoylanilide hydroxamic acid (SAHA) is a hydroxamic acid-containing hybrid polar molecule; SAHA specifically binds to and inhibits the activity of histone deacetylase. Although SAHA, like other inhibitors of histone deacetylase, exhibits antitumor effects by increasing expression of genes regulating tumor survival, we found that SAHA reduces the production of proinflammatory cytokines in vivo and in vitro . A single oral administration of SAHA to mice dose-dependently reduced circulating TNF-α, IL-1-β, IL-6, and IFN-γ induced by lipopolysaccharide (LPS). Administration of SAHA also reduced hepatic cellular injury in mice following i.v. injection of Con A. SAHA inhibited nitric oxide release in mouse macrophages stimulated by the combination of TNF-α plus IFN-γ. Human peripheral blood mononuclear cells stimulated with LPS in the presence of SAHA released less TNF-α, IL-1-β, IL-12, and IFN-γ (50% reduction at 100–200 nM). The production of IFN-γ stimulated by IL-18 plus IL-12 was also inhibited by SAHA (85% at 200 nM). However, SAHA did not affect LPS-induced synthesis of the IL-1-β precursor, the IL-1 receptor antagonist, or the chemokine IL-8. In addition, IFN-γ induced by anti-CD3 was not suppressed by SAHA. Steady-state mRNA levels for LPS-induced TNF-α and IFN-γ in peripheral blood mononuclear cells were markedly decreased, whereas IL-8 and IL-1-β mRNA levels were unaffected. Because SAHA exhibits antiinflammatory properties in vivo and in vitro , inhibitors of histone deacetylase may stimulate the expression of genes that control the synthesis of cytokines and nitric oxide or hyperacetylate other targets.

Purification of proteins synthesised by the solid-phase stepwise approach, using conventional chromatographic tactics is often difficult and results in poor yields. Purification through selective labelling of the target sequence with chromatographic probes when accompanied by an efficient capping protocol is an effective way to drastically improve both the quality and yields of these large, chemically synthesised polypeptides. Basically, the chromatographic probe consists of two parts, an ‘affinity’ group that significantly changes the properties of the peptide possessing it and a reversible linker through which the probe is attached to the peptide. Furthermore, the latter is stable under the conditions used to deprotect and cleave the peptide from the resin support. After separation of labelled sequences from terminated peptides using a suitable ‘affinity’ chromatographic method the label is selectively removed thus yielding the purified polypeptide. Several ‘chromatographic probes’ have been proposed throughout the years. Those based on the FMOC molecule and on the biotin/avidin interaction seem to be of more general applicability and are discussed here.

Leflunomide is one of the most promising disease-modifying antirheumatic drug now in clinical trials for the treatment of rheumatoid arthritis. Metabolic studies have indicated that leflunomide is rapidly processed in vivo to an active metabolite, A771726 (2). To identify the chemical characteristics necessary for the immunosuppressive activity of 2, configurational and conformational studies were carried out on the latter and its inactive analogues (ethyl 3-hydroxy-2-((4-(trifluoromethyl)phenyl)carbamoyl)but-2-enoate, 3a, and 3-hydroxy-2-nitro-N-(4-(trifluoromethyl)phenyl)but-2-enamide, 3b). These studies suggested that the pharmacophore responsible for the immunosuppressive activity of 2 is a beta-keto amide with the enolic hydroxy group cis to the amidic moiety. To verify this hypothesis, a new class of immunosuppressive agents was designed and synthesized. Their testing in vitro and in vivo identified compounds which were more potent than both leflunomide and 2 and above all confirmed our hypothesis as to the key structural and chemical determinants for the immunosuppressive properties of 2 and our compounds.

Organic nitrates have been in therapeutic use for the treatment of angina pectoris for over a century. Recently it was reported that the release of nitric oxide (NO), mediated by a cascade of biochemical events, is responsible for the pharmacological activity of these compounds. The organic nitrates most commonly used, (glyceryl trinitrate (GTN), isosorbide dinitrate (ISDN), isosorbide-5-mononitrate (IS-5-MN) and nicorandil), are useful for the treatment of stable, unstable and Prinzmetal's angina pectoris, for congestive heart failure and myocardial infarction. Nowadays new applications for these compounds, such as ocular hypotension and the protection of gastric mucosa, are emerging. However, the classical organic nitrates are characterized by undesirable side-effects, such as nitrate tolerance, strong_hypotension, reflex tachycardia and headaches. During the last decade, the search for new organic nitrates with reduced side effects and improved oral bioavailability has greatly intensified. The structural modifications studied in order to solve these problems and the structure-activity relationships for each new class of organic nitrates are reported in this review.

The first synthetic pathway to synthesize 6-unsubstituted-2,3-dihydro-1,3-oxazin-4-ones is described. The α-formylation of the starting amide and the cyclization of the α-formylamide with a desired aldehyde under acidic conditions gave compounds 5a−h (R = nPr, iPr, cPr, cHex, Ph, CH2Ph, nHex, and R1 = H, Me). This strategy was used with little modification for the preparation of new monocyclic organic nitrates such as 2a−c (2a (R = Ph, R1 = H, and R2 = H), 2b (R = Ph, R1 = H, and R2 = Me), and 2c (R = H, R1 = Ph, and R2 = H).

The purification of large synthetic peptides using conventional separation techniques often results in poor yields and homogeneity due to the accumulation of chromatographically similar deletion and truncated impurities. We have developed a highly effective synthetic strategy and one-step purification procedure that is based on (i) the application of single coupling using HBTU/HOBt activation to reduce incomplete couplings, (ii) the use of N-(2-chlorobenzyloxycarbonyloxy)succinimide as a capping agent to terminate deletion sequences and (iii) the N-terminal derivatization of the complete peptidyl-resin with a reversible Fmoc-based chromatographic probe possessing enhanced physico-chemical properties (i.e. hydrophobicity, charge or affinity label). We report the application of a biotinylated probe, activated as the succinimidyl carbonate, for the purification of a 101 residue chaperonin protein from Rattus norvegicus (rat cpn10), previously synthesized using an optimized synthetic protocol. Biotinylated rat cpn10 was separated from underivatized impurities on an immobilized monomeric avidin column. Free rat cpn10 was released from avidin-agarose column with 5% aqueous triethylamine and after desalting by RP-HPLC gave 9.9% recovery. Characterization and assessment of homogeneity was achieved using ESI-MS, CZE and RP-HPLC.

The preparation and the physico-chemical characterization of 2H-pyrido[2,3-e]-1,3-oxazine-2,4(3H)-diones, 2H-pyrido[4,3-e]-1,3-oxazine-2,4(3H)-diones, 2H-pyrido[4,3-e]-1,3-oxazin-4(3H)-ones, 2H-thieno[2,3-e]-1,3-oxazin-4(3H)-ones and 2H-thieno[3,4-e]-1,3-oxazine-2,4(3H)-diones are reported.

New nitro ester 3-[(nitrooxy)alkyl]-2H-1,3-benzoxazin-4(3H)-ones show marked inhibitory activity against ischemia-induced electrocardiographic changes, with only limited systemic hemodynamic effects, and are reported in the present study. These new nitro vasodilators are potent inhibitors of the electrocardiographic T-wave and S-T segment elevation induced by intravenous or intracoronary administration of Arg-vasopressin or methacholine in the anesthetized rat. The most active compounds are up to 300- and 600-fold more potent than glyceryl trinitrate or Nicorandil, respectively. These nitro esters relax in a concentration-dependent manner the isolated rabbit aorta, at higher concentrations (2-40-fold) than glyceryl trinitrate, and reduce the mean arterial blood pressure at doses 7-300-fold higher than those required by glyceryl trinitrate to exert a similar hypotensive effect. Remarkably, these compounds retain their anti-ischemic and hemodynamic profile after oral (po) administration. These new nitro ester derivatives, endowed with a marked antianginal activity, which is not associated with concurrent and pronounced falls in systemic blood pressure, represent the leads of a new class of selective nitrovasodilators having a preferential action on large coronary vessels, which could be clinically relevant in the treatment of coronary artery diseases.

Organic nitrates have been in therapeutic use for the treatment of angina pectoris for over a century. During the past decade, the search for new organic nitro esters with reduced side effects and improved oral bioavailability has been greatly intensified. We have discovered a new class of organic nitrates characterized by good oral activity and a coronary vascular selectivity greater than that of glyceryl trinitrate. Full structure-activity relationship studies of this new class of nitroesters are reported. From the screening of these compounds, ITF 296 was chosen for further evaluation.

The preparation and the physico-chemical characterization of new heterocyclic organic nitrates containing 1,3-benzoxazine-2,4-dione, 1,3-benzoxazine-2-thion-4-one, 1,3-benzothiazine-2,4-dione and quinazo-line-2,4-dione moieties, are reported.

The application of reversible Chromatographic probes for the purification of synthetic peptides by reversed-phase (RP) and affinity Chromatography is described. In previous work the concept of a one-step purification procedure based on the acid-stable fluorenylmethoxycarbonyl (Fmoc) group was considered. Similar probes were used to purify peptides from 17 to 104 residues in length, using ion-exchange and RP media. The RP probes described here represent simplified versions of those reported previously. Further, the application of a new probe containing biotin, for affinity Chromatography, is demonstrated. Stepwise solid-phase peptide synthesis was performed using both Boc and Fmoc chemistries and N-(2-chlorobenzyloxycarbonyloxy)succinimide as a capping reagent. The Chromatographic probes were incorporated on to the “target” peptide as active succinimidyl carbonate derivatives. After cleavage using either hydrogen fluoride or trifluoroacetic acid, purification was performed on either RP media or immobilized avidin. Treatment with organic base yielded the free purified product. The efficiency of the new probe molecules and the capping procedure was demonstrated by the purification of two polypeptides, 46 and 101 residues in length.

The 1 H and 13 C NMR of a series of 3-nitrooxyalkyl-2H-1,3-benzoxazin-4-(3H)-ones were measured and assigned on the basis of substituent chemical shifts, DEPT experiments and 13 C/ 1 H shift correlated spectra

The aim of this work was to correlate the in vitro human skin permeability, expressed as the permeability coefficient ( K p ), and some physicochemical parameters of a new series of benzoxazinones. The in vitro human skin permeability of 14 substances, including regioisomers with CH 3 , OH, OCH 3 , and Cl groups in different positions on the aromatic ring, was determined. The modified Franz diffusion cell method was used. The K p values were in the range 0.14–8.24 cm/h, showing a strong dependence on the position and type of substituent. Physicochemical descriptors usually referred in literature, such as log P , molecular weight and volume (MV), hydrogen bond donor ( H d ) and acceptor activity ( H a ), and molecular refractivity were considered, with the addition of solvation enthalpy (ΔΔ H solv ). ΔΔ H solv is defined as the difference between formation enthalpies in water and octanol. The algorithm with the best correlation between K p and physicochemical descriptors was calculated, taking into account the differences observed among the regioisomers. The algorithm obtained with ΔΔ H solv had a good correlation ( r 2 =0.749, F =16.43, P =0.0005), comparable with the equation, proposed by Potts and Guy, based on MV, H d and H a ( r 2 =0.830, F =16.3, P =0.0004).

Publisher Summary This chapter discusses the perspectives in the design and application of dopamine receptor agonists. The chapter discusses main pharmacological and clinical achievements as well as the essential results of the search of pharmacophores and of tentative receptor models, to provide the basis for understanding “third generation” of dopamine receptor agonists. The era of dopamine receptor pharmacology began around 1960. Heralded by the discovery of the neurotransmitter role of dopamine, it was previously regarded only as a metabolic precursor of noradrenaline. A new avenue of investigation of the interactions of agonistic and antagonistic ligands with receptor models directly derived from the structure of the protein is opened by recent advancements. Thus, progress in indirect modeling, based on steric and electronic properties of the ligands, plays an important role not only in the design of new active compounds, but also in the improvement and the validation of the direct models. In these models, it has been generally accepted that at least one phenolic or equivalent function, such as the indole NH group of the ergolines, acts as a hydrogen bond donor with respect to a site of the receptor. The recent discovery of potent dopamine agonists devoid of such functions suggests that recognition modes other than hydrogen bonding may be involved in the corresponding region of the receptor.

Cette synthese est realisee a partir de l'acetal de cetene derive du propionate de N-methylephedrine