Continuous Flow Acylation of (Hetero)aryllithiums with Polyfunctional N,N-Dimethylamides and Tetramethylurea in Toluene.

The continuous flow reaction of various aryl or heteroaryl bromides in toluene in the presence of THF (1.0 equiv) with sec-BuLi (1.1 equiv) provided at 25 °C within 40 sec the corresponding aryllithiums which were acylated with various functionalized N,N-dimethylamides including easily enolizable amides at -20 °C within 27 sec, producing highly functionalized ketones in 48-90 % yield (36 examples). This method was well suited for the preparation of α-chiral ketones such as naproxene and ibuprofen derived ketones with 99 % ee. A one-pot stepwise bis-addition of two different lithium organometallics to 1,1,3,3-tetramethyurea (TMU) provided unsymmetrical ketones in 69-79 % yield (9 examples).

Application of Transition-Metal Catalysis, Biocatalysis, and Flow Chemistry as State-of-the-Art Technologies in the Synthesis of LCZ696.

LCZ696 is a novel treatment for patients suffering from heart failure that combines the two active pharmaceutical ingredients sacubitril and valsartan in a single chemical compound. While valsartan is an established drug substance, a new manufacturing process suitable for large-scale commercial production had to be developed for sacubitril. The use of chemocatalysis, biocatalysis, and flow chemistry as state-of-the-art technologies allowed to efficiently build up the structure of sacubitril and achieve the defined performance targets.

Fouling of Flow Reactors in Organolithium Mediated Transformations: Experience on Scale-up and Proposed Solution.

Continuous processing has been demonstrated to be a superior approach when applied to fast and energetic chemical transformations. Indeed, whereas classical batch or semi-batch methods require cryogenic conditions and slow addition rates of reactive species, flow technologies enable rapid mixing of synthetic partners in a highly controlled environment. As a result, low yielding and dangerous processes in batch can be performed at scale in a cost competitive and safer continuous manner. Despite the advantages of higher quality and safety, the perennial problems of solids build-up and pipe fouling threaten the robustness and reliability of flow processes. In this contribution, a new methodology to prevent reactor fouling is reported and discussed. The implementation of this methodology has been decisive in solving fouling issues encountered during the piloting of an organolithium based flow process.

Investigating the allosteric reverse signalling of PARP inhibitors with microsecond molecular dynamic simulations and fluorescence anisotropy.

The inhibition of the poly(ADP-ribose) polymerase (PARP) family members is a strategy pursued for the development of novel therapeutic agents in a range of diseases, including stroke, cardiac ischemia, cancer, inflammation and diabetes. Even though some PARP-1 inhibitors have advanced to clinical setting for cancer therapy, a great deal of attention is being devoted to understand the polypharmacology of current PARP inhibitors. Besides blocking the catalytic activity, recent works have shown that some PARP inhibitors exhibit a poisoning activity, by trapping the enzyme at damaged sites of DNA and forming cytotoxic complexes. In this study we have used microsecond molecular dynamics to study the allosteric reverse signalling that is at the basis of such an effect. We show that Olaparib, but not Veliparib and HYDAMTIQ, is able to induce a specific conformational drift of the WGR domain of PARP-1, which stabilizes PARP-1/DNA complex through the locking of several salt bridge interactions. Fluorescence anisotropy assays support such a mechanism, providing the first experimental evidence that HYDAMTIQ, a potent PARP inhibitor with neuroprotective properties, is less potent than Olaparib to trap PARP-1/DNA complex.

Glucuronidation of bile acids under flow conditions: design of experiments and Koenigs-Knorr reaction optimization.

An efficient method for the C3-glucuronidation of bile acids is developed under flow conditions. A modular mesoreactor assisted flow set-up was combined with statistical design of experiments to speed up the optimization of the Koenigs-Knorr reaction in terms of yield, regioselectivity, costs, as well as technical and practical standpoints. Using the optimal conditions, selective glucuronidation of naturally occurring bile acids was successfully achieved offering a new, valuable route to C3-glucuronidated bile acids useful for biological, diagnostic and PK/ADMET investigations.

Synthesis and quantitative structure-property relationships of side chain-modified hyodeoxycholic acid derivatives.

Bile acids have emerged as versatile signalling compounds of a complex network of nuclear and membrane receptors regulating various endocrine and paracrine functions. The elucidation of the interconnection between the biological pathways under the bile acid control and manifestations of hepatic and metabolic diseases have extended the scope of this class of steroids for in vivo investigations. In this framework, the design and synthesis of novel biliary derivatives able to modulate a specific receptor requires a deep understanding of both structure-activity and structure-property relationships of bile acids. In this paper, we report the preparation and the critical micellization concentration evaluation of a series of hyodeoxycholic acid derivatives characterized by a diverse side chain length and by the presence of a methyl group at the alpha position with respect to the terminal carboxylic acid moiety. The data collected are instrumental to extend on a quantitative basis, the knowledge of the current structure-property relationships of bile acids and will be fruitful, in combination with models of receptor activity, to design and prioritize the synthesis of novel pharmacokinetically suitable ligands useful in the validation of bile acid-responsive receptors as therapeutic targets.

Extending SAR of bile acids as FXR ligands: discovery of 23-N-(carbocinnamyloxy)-3α,7α-dihydroxy-6α-ethyl-24-nor-5ß-cholan-23-amine.

Within our efforts in the discovery of novel potent and selective ligands for the FXR receptor, 23-N-(carbocinnamyloxy)-3α,7α-dihydroxy-6α-ethyl-24-nor-5ß-cholan-23-amine was synthesized and evaluated for its ability to activate and modulate the biological response of the receptor. Alphascreen and RT-PCR revealed that the 6α-ethyl-24-norcholanyl-23-amine derivate behaves as full FXR agonist endowed with high binding affinity and efficacy, representing a promising lead candidate for further optimization. In addition, docking studies provide new insights into the molecular basis governing the partial and full agonist activity at FXR.

Regioselective difunctionalization of pyridines via 3,4-pyridynes.

A new regioselective 3,4-difunctionalization of 3-chloropyridines via 3,4-pyridyne intermediates is reported. Regioselective lithiation of 3-chloro-2-ethoxypyridine and a related 2-thio-derivative followed by treatment with aryl- and alkylmagnesium halides as well as magnesium thiolates at -78 °C produced 3,4-pyridynes during heating to 75 °C. Regioselective addition of the Grignard moiety in position 4 followed by an electrophilic quench in position 3 led to various 2,3,4-trisubstituted pyridines. This method was adapted into a continuous flow set-up. As an application, we have prepared a key intermediate for (±)-paroxetine.

Intramuscular neridronate in postmenopausal women with low bone mineral density.

Compliance to osteoporosis treatment with oral bisphosphonates is very poor. Intermittent intravenous bisphosphonate is a useful alternative, but this route is not readily available. Neridronate, a nitrogen-containing bisphosphonate that can be given intramuscularly (IM), was tested in a phase 2 clinical trial in 188 postmenopausal osteoporotic women randomized to IM treatment with 25 mg neridronate every 2 weeks, neridronate 12.5 or 25 mg every 4 weeks, or placebo. All patients received calcium and vitamin D supplements. The patients were treated over 12 months with 2-year posttreatment follow-up. After 12-month treatment, all three doses were associated with significant bone mineral density (BMD) increases at both the total hip and spine. A significant dose-response relationship over the three doses was observed for the BMD changes at the total hip but not at the spine. Bone alkaline phosphatase decreased significantly by 40-55% in neridronate-treated patients, with an insignificant dose-response relationship. Serum type I collagen C-telopeptide decreased by 58-79%, with a significant dose-response relationship (P < 0.05). Two years after treatment discontinuation, BMD declined by 1-2% in each dose group, with values still significantly higher than baseline at both the spine and the total hip. Bone turnover markers progressively increased after treatment discontinuation, and on the second year of follow-up the values were significantly higher than pretreatment baseline. The results of this study indicate that IM neridronate might be of value for patients intolerant to oral bisphosphonates and unwilling or unable to undergo intravenous infusion of bisphosphonates.

Discovery of 3α,7α,11ß-Trihydroxy-6α-ethyl-5ß-cholan-24-oic Acid (TC-100), a Novel Bile Acid as Potent and Highly Selective FXR Agonist for Enterohepatic Disorders.

As a continuation of previous efforts in mapping functional hot spots on the bile acid scaffold, we here demonstrate that the introduction of a hydroxy group at the C11ß position affords high selectivity for FXR. In particular, the synthesis and FXR/TGR5 activity of novel bile acids bearing different hydroxylation patterns at the C ring are reported and discussed from a structure-activity standpoint. The results obtained led us to discover the first bile acid derivative endowed with high potency and selectivity at the FXR receptor, 3α,7α,11ß-trihydroxy-6α-ethyl-5ß-cholan-24-oic acid (TC-100, 7) which also shows a remarkable physicochemical and pharmacological profile. Compound 7 combines the excellent physicochemical properties of hydrophilic bile acids such as ursodeoxycholic acid, with the distinct ability to specifically bind and regulate FXR activity in vivo, thus providing a bona fide novel therapeutic agent to treat enterohepatic disorders such as cholestasis, NASH, and inflammatory bowel disease.

Effects of S 21403 on hormone secretion from isolated rat pancreas at different glucose concentrations.

We investigated the in vitro effects of therapeutical concentrations of S 21403 (a succinic acid derivative also known as KAD 1229 and mitiglinide) on insulin and glucagon secretion during a metabolic stimulus (glucose rising from 5 to 8.33 mM) or at a stable 2.22 mM glucose using the isolated perfused rat pancreas model, and we compared them with the patterns of repaglinide and glibenclamide. Control perfusions were also performed. During 8.33 mM glucose, insulin release peaked to 339.12+/-22.87 microU/ml in controls. S 21403 enhanced insulin release (first peak 413.02+/-14.90 microU/ml; P<0.03 vs. controls, P=ns vs. repaglinide, P<0.005 vs. glibenclamide). Repaglinide increased glucose-induced first peak secretion to 409.33+/-20.05 microU/ml within the eighth minute (P<0.05 vs. controls, P<0.01 vs. glibenclamide). Glibenclamide did not affect the first phase of glucose-induced insulin release (peak of 338.41+/-29.79 microU/ml) but potentiated and delayed the second phase. No drug affected glucagon release. In conclusion, S 21403 induces a faster, more physiological pattern of insulin release than the other drugs we tested.

Building a sulfonamide library by eco-friendly flow synthesis.

A rapid and eco-friendly synthesis of a sulfonamide library under flow conditions is described. The study illustrates an efficient, safe, and easily scalable preparation of sulfonamides by use of a meso-reactor apparatus, thus demonstrating the impact of flow technologies within drug discovery. Waste minimization, employment of green media, and nontoxic reactants are achieved by the optimization of the flow setup and experimental protocol designed to sequentially synthesize primary, secondary, and tertiary sulfonamides. Isolation of the products involves only extraction and precipitation affording pure compounds in good to high yields without further purification for biological evaluation.

Bone mineral density in haemophilia patients. A meta-analysis.

Osteoporosis is caused by bone mineral density (BMD) reduction. Haemophilia patients are at increased risk of osteoporosis because of decreased physical activity and blood-borne virus infections. This systematic review of the literature aims at evaluating BMD reduction in severe haemophilia patients and its correlation with patients' characteristics. Seven case-control studies evaluating lumbar BMD values [g/cm2] (all studies), BMI (5/7 studies), and hepatitis C virus (HCV) seropositivity (6/7 studies) in severe haemophilia patients and controls were meta-analysed. Standardised mean difference (SMD) of BMD was used to compare cases and controls. The effect of body mass index (BMI) and HCV infection was investigated by meta-regression. One hundred one adult cases (age 33 +/- 8.9) with 101 controls and 111 paediatric cases (age 8 +/- 3.6) with 307 controls were available for analysis. Lumbar BMD was significantly lower in severe haemophilia patients than in controls, both in adult (pooled SMD -1.379, 95% confidence interval [CI] -2.355 to -0.403, p=0.006) and children (pooled SMD -0.438, 95% CI -0.686 to -0.189, p=0.001). The reduction in BMD in patients versus controls was not significantly correlated with the reduction in BMI or with the percentage of HCV-infected patients. This meta-analysis confirms the association between severe haemophilia and low BMD. Future studies should investigate fracture rates and interventions to prevent bone loss in persons with haemophilia.

Injectable bisphosphonates in the treatment of postmenopausal osteoporosis.

Osteoporosis is a "silent" disease and the patient has usually no clue of it until the occurrence of a fragility fracture. Prevention requires a continuous daily treatment that could be uncomfortable to the patient. Besides the recently introduced weekly oral schedules, injectable bisphosphonates have often been used as an off-label option to ameliorate compliance. In general, although with different efficiency, almost all injectable bisphosphonates can improve bone mineral density and suppress bone resorption markers. The effect of intravenous infusions of bisphosphonates are, to a large extent, similar to equivalent intramuscular administrations, but doses and dosing intervals represent the critical issues. Pain at the injection site and acute phase reactions are relatively common to intramuscular clodronate and intravenous infusions of nitrogen-containing bisphosphonates, respectively. Under certain circumstances, intermittent treatment with injectable bisphosphonates might represent a feasible alternative when compliance is at risk.

The effect of age, weight, and lifestyle factors on calcaneal quantitative ultrasound: the ESOPO study.

Quantitative ultrasound (QUS) techniques have been shown to be as good as bone mineral density (BMD) assessed by dual-energy X-ray absorptiometry (DXA), in predicting fracture risk: QUS technique could increase substantially the accessibility to a reliable bone osteoporosis risk evaluation, but little is know regarding the relationship of QUS to risk factors that have been found to predict DXA-BMD values and this is even more true in men. We studied 6,811 postmenopausal women 40 to 80 years of age and 4,981 men 60-80 years of age representative of the general population of all regions of Italy. All participants were questioned on lifestyle habits and on their medical history. After a physical examination "bone stiffness" (called here for simplicity, stiffness), which is derived from the values of broadband ultrasound attenuation (BUA) and speed of sounds (SoS) was measured by a heel QUS device (Achilles apparatus, Lunar, Madison, USA). The most common recognized determinants of bone mass (either categorical or continuous variables) were modeled with stiffness by multiple regression analysis or ANOVA. Stiffness was strongly related to age and weight. After adjusting for these variables, the women who had taken hormone replacement therapy for more than a year had significantly higher stiffness values. The difference versus nonusers remained significant for up to 20 years-since-menopause (YSM). This effect was so strong that for further analysis these women were excluded. By multivariate analysis, stiffness was then found to be significantly related to recalled body weight at 25 years of age, actual and past cigarettes smoked per day, and dairy calcium intake. Stiffness was also associated with a number of categorial factors adjusted for age, weight, and YSM: prior ovariectomy, history of more than 2 months confined to bed, outdoor physical activity, smoking, chronic use of any drug, and past corticosteroid use. All these categorial and continuous variables predicted stiffness equally in men and women. In conclusion, QUS bone measurements discriminate postmenopausal women according to past use of hormone replacement therapy. Risk factors usually associated to BMD as measured by DXA are also associated to calcaneal bone stiffness as measured by QUS, and most risk factors for osteoporosis usually observed in women are equally applicable to men.

Effects of high dose methylprednisolone pulse therapy on bone mass and biochemical markers of bone metabolism in patients with active rheumatoid arthritis: a 12-month randomized prospective controlled study.

OBJECTIVE: To study the effects of one year of high dose 6-methylprednisolone pulse therapy (MPPT) on bone mass, seric bone alkaline phosphatase (sBAP), and urinary deoxypyridinoline (uDpyr) in patients with active rheumatoid arthritis (RA), and to compare results with those of patients with active RA treated with oral methylprednisolone (OMP). METHODS: Thirty-one women with active RA were given 1000 mg of MP IV for 3 alternate days, with a mean interval of administration of 76 days (+/- 8.3 SD) for one year (MPPT group). Bone mineral density (BMD) (total body, lumbar spine, and femur neck), plasma levels of sBAP, and urinary concentrations of uDpyr were assessed at the beginning of the treatment and every 3 months until the end of the study. Moreover, erythrocyte sedimentation rate (ESR), Thompson joint score, and early morning stiffness were assessed at study entry and every month. The control group, 31 women with active RA treated with oral MP, was followed in the same way (OMP group). RESULTS: In the MPPT group there was no significant reduction of BMD at any site compared to significant reductions in lumbar BMD at 6 and 12 months and total body BMD and femur neck BMD at 12 months in the OMP group. Also in the OMP group, a significant reduction in the mean sBAP was observed. The mean uDpyr levels were not significantly reduced in either group. CONCLUSION: Our results show that MPPT, compared to continuous therapy with oral corticosteroids, preserves bone mass without modifying the biochemical markers of bone metabolism.