PDE4 Inhibitors, Boehringer Ingelheim Pharmaceuticals

PRECLINICAL Comments Off

May 182016

R CONF SHOWN

BI ?

(R)-2-(4-(4-Chlorophenoxy)piperidin-1-yl)-4-((tetrahydro-2H-pyran-4-yl)amino)-6,7-dihydrothieno[3,2-d]pyrimidine 5-Oxide

C₂₂ H₂₇ Cl N₄ O₃ S, 462.99

CAS 1910076-27-5

Thieno[3,2-d]pyrimidin-4-amine, 2-[4-(4-chlorophenoxy)-1-piperidinyl]-6,7-dihydro-N-(tetrahydro-2H-pyran-4-yl)-, 5-oxide, (5R)-

¹H NMR (400 MHz, CDCl₃) δ 1.49 (dq, J = 4.2, 11.8 Hz, 1H), 1.62 (dq, J = 4.2, 11.8 Hz, 1H), 1.74–1.89 (m, 3H), 1.90–2.02 (m, 3H), 2.96–3.07 (m, 2H), 3.29 (dt, J = 13.6, 8.4 Hz, 1H), 3.44 (ddd, J = 19.2, 11.2, 2.0 Hz, 2H), 3.62 (dt, J = 17.2, 7.8 Hz, 1H), 3.76 (m, 2H), 3.96 (dd, J = 15.6, 12.8 Hz, J = 2H), 4.09–3.99 (m, 3H), 4.51 (m, 1H), 6.21 (br d, J = 6.0 Hz, 1H), 6.86 (d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.8 Hz, 2H);

¹³C NMR (100 MHz, CDCl₃) δ 30.4, 32.5, 32.7, 41.0, 47.2, 49.6, 66.9, 66.9, 72.9, 107.8, 117.5, 125.9, 129.5, 155.8, 158.9, 163.0, 174.6.

The use of phosphodiesterase type 4 (PDE4) inhibitors for the treatment of COPD (chronic obstructive pulmonary disease) by reducing inflammation and improving lung function is well documented. Given the potential therapeutic benefit offered by these compounds, a number of PDE4-selective inhibitors containing a dihydrothieno[3,2-d]pyrimidine core were identified as preclinical candidates in Boehringer Ingelheim Pharmaceuticals discovery laboratories

While the pathogenesis of chronic obstructive pulmonary disease (COPD) is incompletely understood, chronic inflammation is a major factor. In fact, the inflammatory response is abnormal, with CD8⁺ T-cells, CD68⁺ macrophages, and neutrophils predominating in the conducting airways, lung parenchyma, and pulmonary vasculature. Elevated levels of the second messenger cAMP can inhibit some inflammatory processes. Theophylline has long been used in treating asthma; it causes bronchodilation by inhibiting cyclic nucleotide phosphodiesterase (PDE), which inactivates cAMP. By inhibiting PDE, theophylline increases cAMP, inhibiting inflammation and relaxing airway smooth muscle. Rather than one PDE, there are now known to be more than 50, with differing activities, substrate preferences, and tissue distributions. Thus, the possibility exists of selectively inhibiting only the enzyme(s) in the tissue(s) of interest. PDE 4 is the primary cAMP-hydrolyzing enzyme in inflammatory and immune cells (macrophages, eosinophils, neutrophils). Inhibiting PDE 4 in these cells leads to increased cAMP levels, down-regulating the inflammatory response. Because PDE 4 is also expressed in airway smooth muscle and, in vitro, PDE 4 inhibitors relax lung smooth muscle, selective PDE 4 inhibitors are being developed for treating COPD. Clinical studies have been conducted with PDE 4 inhibitors;

Chronic obstructive pulmonary disease (COPD) is a serious and increasing global public health problem; physiologically, it is characterized by progressive, irreversible airflow obstruction and pathologically, by an abnormal airway inflammatory response to noxious particles or gases (MacNee 2005a). The COPD patient suffers a reduction in forced expiratory volume in 1 second (FEV₁), a reduction in the ratio of FEV₁ to forced vital capacity (FVC), compared with reference values, absolute reductions in expiratory airflow, and little improvement after treatment with an inhaled bronchodilator. Airflow limitation in COPD patients results from mucosal inflammation and edema, bronchoconstriction, increased secretions in the airways, and loss of elastic recoil. Patients with COPD can experience ‘exacerbations,’ involving rapid and prolonged worsening of symptoms (Seneff et al 1995; Connors et al 1996; Dewan et al 2000; Rodriguez-Roisin 2006; Mohan et al 2006). Many are idiopathic, though they often involve bacteria; airway inflammation in exacerbations can be caused or triggered by bacterial antigens (Murphy et al 2000; Blanchard 2002; Murphy 2006;Veeramachaneni and Sethi 2006). Increased IL-6, IL-1β, TNF-α, GRO-α, MCP-1, and IL-8 levels are found in COPD patient sputum; their levels increase further during exacerbations. COPD has many causes and significant differences in prognosis exist, depending on the cause (Barnes 1998; Madison and Irwin 1998).

COPD is already the fourth leading cause of death worldwide, according to the World Health Organization (WHO); the WHO estimates that by the year 2020, COPD will be the third-leading cause of death and the fifth-leading cause of disability worldwide (Murray and Lopez 1997). COPD is the fastest-growing cause of death in developed nations and is responsible for over 2.7 million deaths per year worldwide. In the US, there are currently estimated to be 16 million people with COPD. There are estimated to be up to 20 million sufferers in Japan, which has the world’s highest per capita cigarette consumption and a further 8–12 million in Europe. In 2000, COPD accounted for over 20 million outpatient visits, 3.4 million emergency room visits, 6 million hospitalizations, and 116,500 deaths in the US (National Center for Health Statistics 2002). Factors associated with COPD, including immobility, often lead to secondary health consequences (Polkey and Moxham 2006).

Risk factors for the development of COPD include cigarette smoking, and occupational exposure to dust and chemicals (Senior and Anthonisen 1998; Anthonisen et al 2002; Fabbri and Hurd 2003; Zaher et al 2004). Smoking is the most common cause of COPD and the underlying inflammation typically persists in ex-smokers. Oxidative stress from cigarette smoke is also an issue in COPD (Domej et al 2006). Despite this, relatively few smokers ever develop COPD (Siafakas and Tzortzaki 2002).

While many details of the pathogenesis of COPD remain unclear, chronic inflammation is now recognized as a major factor, predominantly in small airways and lung parenchyma, characterized by increased numbers of macrophages, neutrophils, and T-cells (Barnes 2000; Stockley 2002). As recently as 1995, the American Thoracic Society issued a statement defining COPD without mentioning the underlying inflammation (American Thoracic Society 1995). Since then, the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines have made it clear that chronic inflammation throughout the airways, parenchyma, and pulmonary vasculature plays a central role (Pauwels et al 2001; GOLD 2003). The comparatively recent realization of the role of airway inflammation in COPD has altered thinking with regard to potential therapies (Rogers and Giembycz 1998; Vignola 2004).

Most pharmacological therapies available for COPD, including bronchodilator and anti-inflammatory agents, were first developed for treating asthma. The mainstays of COPD treatment are inhaled corticosteroids (McEvoy and Niewoehner 1998; Borron and deBoisblanc 1998; Pauwels 2002; Gartlehner et al 2006;D’Souza 2006), supplemental oxygen (Petty 1998; Austin and Wood-Baker 2006), inhaled bronchodilators (Costello 1998; Doherty and Briggs 2004), and antibiotics (Taylor 1998), especially in severely affected patients (Anthonisen et al 1987; Saint et al 1995; Adams et al 2001; Miravitlles et al 2002; Donnelly and Rogers 2003; Sin et al 2003; Rabe 2006), though the use of antibiotics remains controversial (Ram et al 2006). Long-acting β₂-agonists (LABAs) improve the mucociliary component of COPD. Combination therapy with LABAs and anticholinergic bronchodilators resulted in modest benefits and improved health-related quality of life (Buhl and Farmer 2005; Appleton et al 2006). Treatment with mucolytics reduced exacerbations and the number of days of disability (Poole and Black 2006). The combined use of inhaled corticosteroids and LABAs has been demonstrated to produce sustained improvements in FEV₁ and positive effects on quality of life, number of hospitalizations, distance walked, and exacerbations (Mahler et al 2002;Szafranski et al 2003; Sin et al 2004; Miller-Larsson and Selroos 2006; van Schayck and Reid 2006). However, all of these treatments are essentially palliative and do not impact COPD progression (Hay 2000;Gamble et al 2003; Antoniu 2006a).

A further complication in drug development and therapy is that it can be difficult to determine the efficacy of therapy, because COPD has a long preclinical stage, is progressive, and patients generally do not present for treatment until their lung function is already seriously impaired. Moreover, because COPD involves irreversible loss of elasticity, destruction of the alveolar wall, and peribronchial fibrosis, there is often little room for clinical improvement.

Smoking cessation remains the most effective intervention for COPD. Indeed, to date, it is the only intervention shown to stop the decline in lung function, but it does not resolve the underlying inflammation, which persists even in ex-smokers. Smoking cessation is typically best achieved by a multifactor approach, including the use of bupropion, a nicotine replacement product, and behavior modification (Richmond and Zwar 2003).

In COPD, there is an abnormal inflammatory response, characterized by a predominance of CD8⁺ T-cells, CD68⁺ macrophages, and neutrophils in the conducting airways, lung parenchyma, and pulmonary vasculature (Soto and Hanania 2005; O’Donnell et al 2006; Wright and Churg 2006). Inflammatory mediators involved in COPD include lipids, inflammatory peptides, reactive oxygen and nitrogen species, chemokines, cytokines, and growth factors. COPD pathology also includes airway remodeling and mucociliary dysfunction (mucus hypersecretion and decreased mucus transport). Corticosteroids reduce the number of mast cells, but CD8⁺ and CD68⁺ cells, and neutrophils, are little affected (Jeffery 2005). Inflammation in COPD is not suppressed by corticosteroids, consistent with it being neutrophil-, not eosinophil-mediated. Corticosteroids also do not inhibit the increased concentrations of IL-8 and TNF-α (both neutrophil chemoattractants) found in induced sputum from COPD patients. Neutrophil-derived proteases, including neutrophil elastase and matrix metalloproteinases (MMPs), are involved in the inflammatory process and are responsible for the destruction of elastin fibers in the lung parenchyma (Mercer et al 2005; Gueders et al 2006). MMPs play important roles in the proteolytic degradation of extracellular matrix (ECM), in physiological and pathological processes (Corbel, Belleguic et al 2002). PDE 4 inhibitors can reduce MMP activity and the production of MMPs in human lung fibroblasts stimulated with pro-inflammatory cytokines (Lagente et al 2005). In COPD, abnormal remodeling results in increased deposition of ECM and collagen in lungs, because of an imbalance of MMPs and TIMPs (Jeffery 2001). Fibroblast/myofibroblast proliferation and activation also occur, increasing production of ECM-degrading enzymes (Crouch 1990; Segura-Valdez et al 2000). Additionally, over-expression of cytokines and growth factors stimulates lung fibroblasts to synthesize increased amounts of collagen and MMPs, including MMP-1 (collagenase-1) and MMP-2 and MMP-9 (gelatinases A and B) (Sasaki et al 2000; Zhu et al 2001).

It is now generally accepted that bronchial asthma is also a chronic inflammatory disease (Barnes et al 1988;Barnes 1995). The central role of inflammation of the airways in asthma’s pathogenesis is consistent with the efficacy of corticosteroids in controlling clinical symptoms. Eosinophils are important in initiating and continuing the inflammatory state (Holgate et al 1987; Bruijnzeel 1989; Underwood et al 1994; Teixeira et al 1997), while other inflammatory cells, including lymphocytes, also infiltrate the airways (Holgate et al 1987;Teixeira et al 1997). The familiar acute symptoms of asthma are the result of airway smooth muscle contraction. While recognition of the key role of inflammation has led to an emphasis on anti-inflammatory therapy in asthma, a significant minority of patients remains poorly controlled and some exhibit accelerated declines in lung function, consistent with airway remodeling (Martin and Reid 2006). Reversal or prevention of structural changes in remodeling may require additional therapy (Burgess et al 2006).

There is currently no cure for asthma; treatment depends primarily on inhaled glucocorticoids to reduce inflammation (Taylor 1998; Petty 1998), and inhaled bronchodilators to reduce symptoms (Torphy 1994;Costello 1998; Georgitis 1999; DeKorte 2003). Such treatments, however, do not address disease progression.

COPD and asthma are both characterized by airflow obstruction, but they are distinct in terms of risk factors and clinical presentation. While both involve chronic inflammation and cellular infiltration and activation, different cell types are implicated and there are differences in the inflammatory states (Giembycz 2000;Fabbri and Hurd 2003; Barnes 2006). In COPD, neutrophil infiltration into the airways and their activation appear to be key (Stockley 2002); in asthma, the inflammatory response involves airway infiltration by activated eosinophils and lymphocytes, and T-cell activation of the allergic response (Holgate et al 1987;Saetta et al 1998; Barnes 2006). While macrophages are present in both conditions, the major controller cells are CD8⁺ T-cells in COPD (O’Shaughnessy et al 1997; Saetta et al 1998) and CD4⁺ T-cells in asthma. IL-1, IL-8, and TNF-α are the key cytokines in COPD, while in asthma, IL-4, IL-5, and IL-13 are more important. There are differences in histopathological features of lung biopsies between COPD patients and asthmatics; COPD patients have many fewer eosinophils in lung tissue than asthmatics.

While the early phases of COPD and asthma are distinguishable, there are common features, including airway hyper-responsiveness and mucus hypersecretion. MUC5AC is a major mucin gene expressed in the airways; its expression is increased in COPD and asthmatic patients. At least in vitro, epidermal growth factor stimulates MUC5AC mRNA and protein expression; this can be reversed by PDE 4 inhibitors, which may contribute to their clinical efficacy in COPD and asthma (Mata et al 2005). Similar structural and fibrotic changes make COPD and asthma much less distinguishable in extreme cases; the chronic phases of both involve inflammatory responses, alveolar detachment, mucus hypersecretion, and subepithelial fibrosis. The two conditions have been linked epidemiologically; adults with asthma are up to 12 times more likely to develop COPD over time than those without (Guerra 2005).

PAPER

A practical, safe, and efficient process for the synthesis of PDE4 (phosphodiesterase type 4) inhibitors represented by 1 and 2 was developed and demonstrated on a multi-kilogram scale. Key aspects of the process include the regioselective synthesis of dihydrothieno[3,2-d]pyrimidine-2,4-diol 9 and the asymmetric sulfur oxidation of intermediate 11.

Development of a Practical Process for the Synthesis of PDE4 Inhibitors

Rogelio P. Frutos^*, ET AL

Chemical Development US, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, P.O. Box 368, Ridgefield, Connecticut 06877-0368, United States

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.6b00104

http://pubs.acs.org/doi/full/10.1021/acs.oprd.6b00104

PDE 4 in COPD

With regard to COPD, PDE 4 is the primary cAMP-hydrolyzing enzyme in inflammatory and immune cells, especially macrophages, eosinophils, and neutrophils, all of which are found in the lungs of COPD and asthma patients (Torphy et al 1992; Karlsson and Aldous 1997; De Brito et al 1997; Wang et al 1999;Torphy and Page 2000). Inhibition of PDE 4 leads to elevated cAMP levels in these cells, down-regulating the inflammatory response (Dyke and Montana 2002).

PDE 4 has also attracted much attention because it is expressed in airway smooth muscle (Ashton et al 1994;Undem et al 1994; Nicholson et al 1995; Kerstjens and Timens 2003; Mehats et al 2003; Lipworth 2005; Fan Chung 2006). In vitro, PDE 4 inhibitors relax lung smooth muscle (Undem et al 1994; Dent and Giembycz 1995). In COPD and asthma, a selective PDE 4 inhibitor with combined bronchodilatory and anti-inflammatory properties would seem desirable (Nicholson and Shahid 1994; Lombardo 1995; Palfreyman 1995; Cavalia and Frith 1995; Palfreyman and Souness 1996; Karlsson and Aldous 1997; Compton et al 2001; Giembycz 2002; Jacob et al 2002; Soto and Hanania 2005).

PDE 4 inhibitors in COPD

So, because PDE 4 inhibitors suppress inflammatory functions in several cell types involved in COPD and asthma (Huang and Mancini 2006) and because, at least in vitro, PDE 4 inhibitors relax lung smooth muscle, selective PDE 4 inhibitors, originally intended for use in treating depression (Renau 2004), have been developed for the treatment of COPD and asthma (Torphy et al 1999; Spina 2000; Huang et al 2001; Spina 2004; Giembycz 2005a, 2005b; Lagente et al 2005; Boswell-Smith, Spina et al 2006). PDE 4 enzymes are strongly inhibited by the antidepressant drug rolipram (Pinto et al 1993), which decreases the influx of inflammatory cells at sites of inflammation (Lagente et al 1994; Lagente et al 1995; Alves et al 1996). PDE 4 inhibitors down-regulate cytokine production in inflammatory cells, in vivo and in vitro (Undem et al 1994;Dent and Giembycz 1995). TNF-α is an important inflammatory cytokine in COPD; its release is reduced by PDE 4 inhibitors (Souness et al 1996; Chambers et al 1997; Griswold et al 1998; Gonçalves de Moraes et al 1998; Corbel, Belleguic et al 2002). Some PDE 4 inhibitors, including cilomilast and AWD 12-281, can inhibit neutrophil degranulation, a property not shared by theophylline (Ezeamuzie 2001; Jones et al 2005). PDE 4 inhibitors reduce overproduction of other pro-inflammatory mediators, including arachidonic acid and leukotrienes (Torphy 1998). PDE 4 inhibitors also inhibit cellular trafficking and microvascular leakage, production of reactive oxygen species, and cell adhesion molecule expression in vitro and in vivo (Sanz et al 2005). PDE 4 inhibitors, including cilomilast and CI-1044, inhibit LPS-stimulated TNF-α production in whole blood from COPD patients (Burnouf et al 2000; Ouagued et al 2005).

There are now thought to be at least four PDE 4s, A, B, C, and D, derived from four genes (Lobbam et al 1994; Muller et al 1996; Torphy 1998; Conti and Jin 1999; Matsumoto et al 2003). Alternative splicing and alternative promoters add further complexity (Manganiello et al 1995; Horton et al 1995; Torphy 1998). Indeed, the four genes encode more than 16 PDE 4 isoforms, which can be divided into short (∼65–75 kDa) and long forms (∼80–130 kDa); the difference between the short and long forms lies in the N-terminal region (Bolger et al 1997; Huston et al 2006). PDE 4 isoforms are regulated by extracellular signal-related protein kinase (ERK), which can phosphorylate PDE 4 (Houslay and Adams 2003).

The four PDE 4 genes are differentially expressed in various tissues (Silver et al 1988; Lobbam et al 1994;Manganiello et al 1995; Horton et al 1995; Muller et al 1996; Torphy 1998). PDE 4A is expressed in many tissues, but not in neutrophils (Wang et al 1999). PDE 4B is also widely expressed and is the predominant PDE 4 subtype in monocytes and neutrophils (Wang et al 1999), but is not found in cortex or epithelial cells (Jin et al 1998). Upregulation of the PDE 4B enzyme in response to pro-inflammatory agents suggest that it has a role in inflammatory processes (Manning et al 1999). PDE 4C is expressed in lung and testis, but not in circulating inflammatory cells, cortex, or hippocampus (Obernolte et al 1997; Manning et al 1999; Martin-Chouly et al 2004). PDE 4D is highly expressed in lung, cortex, cerebellum, and T-cells (Erdogan and Houslay 1997; Jin et al 1998). PDE 4D also plays an important role in airway smooth muscle contraction (Mehats et al 2003).

A major issue with early PDE 4 inhibitors was their side effect profile; the signature side effects are largely gastrointestinal (nausea, vomiting, increased gastric acid secretion) and limited the therapeutic use of PDE 4 inhibitors (Dyke and Montana 2002). The second generation of more selective inhibitors, such as cilomilast and roflumilast, have improved side effect profiles and have shown clinical efficacy in COPD and asthma (Barnette 1999; Spina 2000; Lagente et al 2005). However, even cilomilast and roflumilast, the most advanced clinical candidates, discussed below, cause some degree of emesis (Spina 2003).

It is now thought that the desirable anti-inflammatory properties and unwanted side effects of nausea and emesis are associated with distinct biochemical activities (Torphy et al 1992; Jacobitz et al 1996; Barnette et al 1996; Souness et al 1997; Souness and Rao 1997). Specifically, the side effects are believed to be associated with the so-called ‘high-affinity rolipram binding site’ (HARBS) (Barnette et al 1995; Muller et al 1996; Jacobitz et al 1996; Kelly et al 1996; Torphy 1998) and/or inhibition of the form of PDE 4 found in the CNS (Barnette et al 1996). The exact nature of HARBS remains unclear, although it has been described as a conformer of PDE 4 (Souness and Rao 1997; Barnette et al 1998). Using mice deficient in PDE 4B or PDE 4D, it appears that emesis is the result of selective inhibition of PDE 4D (Robichaud et al 2002; Lipworth 2005), which is unfortunate, because the most clinically advanced PDE 4 inhibitors are selective for PDE 4D. Also, from animal studies, it appears that the nausea and vomiting are produced via the CNS, though there may also be direct effects on the gastrointestinal system (Barnette 1999).

While beyond the scope of this review, it has been proposed that PDE 4 inhibitors may be useful in treating inflammatory bowel disease (Banner and Trevethick 2004), cystic fibrosis (Liu et al 2005), pulmonary arterial hypertension (Growcott et al 2006), myeloid and lymphoid malignancies (Lerner and Epstein 2006), Alzheimer’s disease (Ghavami et al 2006), rheumatoid arthritis and multiple sclerosis (Dyke and Montana 2002), infection-induced preterm labor (Oger et al 2004), depression (Wong et al 2006), and allergic disease (Crocker and Townley 1999). Varying degrees of in vitro, in vivo, and clinical data exist to support these claims.

So, after that theoretical buildup, we reach the proof of the pudding; clinical studies have been conducted with PDE 4 inhibitors. A potent, but not-very-selective, PDE 4 inhibitor is approved in Japan and is used clinically, including for treating asthma. Another is awaiting approval in the US. One is in advanced clinical development and others are at earlier stages.

REF

Pouzet, P.; Hoenke, C.; Martyres, D.; Nickolaus, P.; Jung, B.; Hamman, H. Dihydrothienopyrimidines for the treatment of inflammatory diseases. PatentWO 2006111549 A1, October 26, 2006.

Ohnacker, G.; Woitun, E. Novel dihydrothieno[3, 2-d]pyrimidines. U.S. Patent US 3,318,881, May 9, 1967.

/////PDE4 Inhibitors, Boehringer Ingelheim Pharmaceuticals, BI ?, PRECLINICAL, 1910076-27-5

Supporting Info

Clc1ccc(cc1)OC2CCN(CC2)c4nc(NC3CCOCC3)c5c(n4)CCS5=O

BMS-520, a Potent and Selective Isoxazole-Containing S1P1 Receptor Agonist

PRECLINICAL, Uncategorized Comments Off

May 132016

BMS-520
CAS: 1236188-38-7
MF: C23H17F3N4O4
MW: 470.1202

Synonym: BMS-520; BMS 520; BMS520.

INNOVATOR Bristol-Myers Squibb Company

INVENTORS

Scott Hunter Watterson, Alaric J. Dyckman,William J. Pitts, Steven H. Spergel

1-[4-[5-[3-Phenyl-4-(trifluoromethyl)isoxazol-5-yl]-1,2,4-oxadiazol-3-yl]benzyl]azetidine-3-carboxylic acid

1-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid

US2011300165

¹H NMR (500 MHz, DMSO-d₆) δ: 3.20–3.46 (m, 5H), 3.66 (s, 2H), 7.53 (d, J = 8.25 Hz, 2H), 7.60–7. 70 (m, 5H), and 8.06 (d, J = 7. 70 Hz, 2H);

MS m/e 471(M+H⁺);

HPLC (XBridge 5 μ C18 4.6 × 50 mm, 4 mL/min, solvent A: 10% MeOH/water with 0.2% H₃PO₄, solvent B: 90% MeOH/water with 0.2% H₃PO₄, gradient with 0–100% B over 4 min): 3.14 min;

Anal. Calcd for C₂₃H₁₇N₄O₄F₃•0.01 EtOH: C, 58.72; H, 3.65; N, 11.90. Found: C, 58.63; H, 3.41; N, 11.84.

BMS-520 is a potent and selective S1P1 agonist. BMS-520 demonstrated impressive efficacy when administered orally in a rat model of arthritis and in a mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Agonism of S1P1, in particular, has been shown to play a significant role in lymphocyte trafficking from the thymus and secondary lymphoid organs, resulting in immunosuppression.

Sphingosine-1 -phosphate (SlP) has been demonstrated to induce many cellular effects, including those that result in platelet aggregation, cell proliferation, cell morphology, tumor cell invasion, endothelial cell and leukocyte chemotaxis, endothelial cell in vitro angiogenesis, and lymphocyte trafficking. SlP receptors are therefore good targets for a wide variety of therapeutic applications such as tumor

15 growth inhibition, vascular disease, and autoimmune diseases. SlP signals cells in part via a set of G protein-coupled receptors named SlPi or SlPl, SIP₂ or S1P2, SIP3 or S1P3, SlP₄ Or S1P4, and SlP₅ or S1P5 (formerly called EDG-I, EDG-5, EDG-3, EDG-6, and EDG-8, respectively). [0003] SlP is important in the entire human body as it is also a major regulator of

20 the vascular and immune systems. In the vascular system, SlP regulates angiogenesis, vascular stability, and permeability. In the immune system, SlP is recognized as a major regulator of trafficking of T- and B-cells. SlP interaction with its receptor SlPi is needed for the egress of immune cells from the lymphoid organs (such as thymus and lymph nodes) into the lymphatic vessels. Therefore, modulation

25 of SlP receptors was shown to be critical for immunomodulation, and SlP receptor modulators are novel immunosuppressive agents.

The SlPi receptor is expressed in a number of tissues. It is the predominant family member expressed on lymphocytes and plays an important role in lymphocyte trafficking. Downregulation of the SlPi receptor disrupts lymphocyte

30 migration and homing to various tissues. This results in sequestration of the lymphocytes in lymph organs thereby decreasing the number of circulating lymphocytes that are capable of migration to the affected tissues. Thus, development of an SlPi receptor agent that suppresses lymphocyte migration to the target sites associated with autoimmune and aberrant inflammatory processes could be efficacious in a number of autoimmune and inflammatory disease states. [0005] Among the five SlP receptors, SlPi has a widespread distribution and is highly abundant on endothelial cells where it works in concert with S IP3 to regulate cell migration, differentiation, and barrier function. Inhibition of lymphocyte recirculation by non-selective SlP receptor modulation produces clinical immunosuppression preventing transplant rejection, but such modulation also results in transient bradycardia. Studies have shown that SlPi activity is significantly correlated with depletion of circulating lymphocytes. In contrast, SIP₃ receptor agonism is not required for efficacy. Instead, SIP3 activity plays a significant role in the observed acute toxicity of nonselective SlP receptor agonists, resulting in the undesirable cardiovascular effects, such as bradycardia and hypertension. (See, e.g., Hale et al, Bioorg. Med. Chem. Lett., 14:3501 (2004); Sanna et al, J. Biol. Chem., 279: 13839 (2004); Anliker et al., J. Biol. Chem., 279:20555 (2004); Mandala et al., J. Pharmacol. Exp. Ther., 309:758 (2004).)

An example of an SlPi agonist is FTY720. This immunosuppressive compound FTY720 (JPI 1080026-A) has been shown to reduce circulating lymphocytes in animals and humans, and to have disease modulating activity in animal models of organ rejection and immune disorders. The use of FTY720 in humans has been effective in reducing the rate of organ rejection in human renal transplantation and increasing the remission rates in relapsing remitting multiple sclerosis (see Brinkman et al., J. Biol. Chem., 277:21453 (2002); Mandala et al., Science, 296:346 (2002); Fujino et al., J. Pharmacol, and Exp. Ther., 305:45658 (2003); Brinkman et al., Am. J. Transplant, 4: 1019 (2004); Webb et al., J.

Neuroimmunol, 153: 108 (2004); Morris et al., Eur. J. Immunol, 35:3570 (2005); Chiba, Pharmacology & Therapeutics, 108:308 (2005); Kahan et al., Transplantation, 76: 1079 (2003); and Kappos et al., N. Engl. J. Med, 335: 1124 (2006)). Subsequent to its discovery, it has been established that FTY720 is a prodrug, which is phosphorylated in vivo by sphingosine kinases to a more biologically active agent that has agonist activity at the SlPi, SIP3, SlP₄, and SIP5 receptors. It is this activity on the SlP family of receptors that is largely responsible for the pharmacological effects of FTY720 in animals and humans.

Clinical studies have demonstrated that treatment with FTY720 results in bradycardia in the first 24 hours of treatment (Kappos et al., N. Engl. J. Med., 335: 1124 (2006)). The observed bradycardia is commonly thought to be due to agonism at the SIP₃ receptor. This conclusion is based on a number of cell based and animal experiments. These include the use of SIP3 knockout animals which, unlike wild type mice, do not demonstrate bradycardia following FTY720 administration and the use of SlPi selective compounds. (Hale et al., Bioorg. Med. Chem. Lett., 14:3501 (2004); Sanna et al., J. Biol. Chem., 279: 13839 (2004); and Koyrakh et al., Am. J. Transplant., 5:529 (2005)).

The following applications have described compounds as SlPi agonists: WO 03/061567 (U.S. Publication No. 2005/0070506), WO 03/062248 (U.S. Patent No. 7,351,725), WO 03/062252 (U.S. Publication No. 2005/0033055), WO 03/073986 (U.S. Patent No. 7,309,721), WO 03/105771, WO 05/058848, WO

06/047195, WO 06/100633, WO 06/115188, WO 06/131336, WO 2007/024922, WO 07/116866, WO 08/023783 (U.S. Publication No. 2008/0200535), and WO 08/074820. Also see Hale et al., J. Med. Chem., 47:6662 (2004). [0009] There still remains a need for compounds useful as SlPi agonists and yet having selectivity over Sl P3.

BMS 520

Paper

Journal of Medicinal Chemistry (2016), 59(6), 2820-2840

Potent and Selective Agonists of Sphingosine 1-Phosphate 1 (S1P₁): Discovery and SAR of a Novel Isoxazole Based Series

Bristol-Myers Squibb Research and Development, P.O. Box 4000, Princeton, New Jersey 08543, United States

J. Med. Chem., 2016, 59 (6), pp 2820–2840

DOI: 10.1021/acs.jmedchem.6b00089

Publication Date (Web): February 28, 2016

*Phone: 609-252-6778. E-mail: scott.watterson@bms.com.

Abstract

Sphingosine 1-phosphate (S1P) is the endogenous ligand for the sphingosine 1-phosphate receptors (S1P_1–5) and evokes a variety of cellular responses through their stimulation. The interaction of S1P with the S1P receptors plays a fundamental physiological role in a number of processes including vascular development and stabilization, lymphocyte migration, and proliferation. Agonism of S1P₁, in particular, has been shown to play a significant role in lymphocyte trafficking from the thymus and secondary lymphoid organs, resulting in immunosuppression. This article will detail the discovery and SAR of a potent and selective series of isoxazole based full agonists of S1P₁. Isoxazole 6d demonstrated impressive efficacy when administered orally in a rat model of arthritis and in a mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis.

SEE…..http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.6b00089

PAPER

This article reports an efficient scale-up synthesis of 1-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)-1,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid (BMS-520), a potent and selective isoxazole-containing S1P₁ receptor agonist. This process features a highly regioselective cycloaddition leading to a key intermediate, ethyl 3-phenyl-4-(trifluoromethyl)isoxazole-5-carboxylate, a chemo-selective hydrolysis of its regioisomers, as well as an improved method for 1,2,4-oxadiazole formation, relative to the original synthesis. The improved process was applied to the preparation of multiple batches of BMS-520 for preclinical toxicological studies.

An Efficient Scale-Up Synthesis of BMS-520, a Potent and Selective Isoxazole-Containing S1P₁ Receptor Agonist

Xiaoping Hou^*, Juliang Zhu, Bang-Chi Chen, Scott H. Watterson, William J. Pitts, Alaric J. Dyckman, Percy H. Carter, Arvind Mathur, and Huiping Zhang

Discovery Chemistry, Bristol-Myers Squibb Research and Development, Route 206 and Provinceline Road, Princeton, New Jersey 08543, United States

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.6b00112

Publication Date (Web): May 05, 2016

*xiaoping.hou@bms.com.

SEE……http://pubs.acs.org/doi/abs/10.1021/acs.oprd.6b00112

.HPLC purity 99.8%; t_R= 7.62 min (method A); 99.9%; t_R = 8.45 min (method B);

LCMS (ESI) m/z calcd for C₂₃H₁₇F₃N₄O₄ [M + H]⁺ 445.2. Found: 471.3.

¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.20–3.46 (m, 5H), 3.66 (s, 2H), 7.53 (d, J = 8.25 Hz, 2H), 7.60–7.70 (m, 5H), and 8.06 (d, J = 7.70 Hz, 2H).

Anal. Calcd for C₂₃H₁₇N₄O₄F₃, 0.44% water: C, 58.42; H, 3.70; N, 11.83. Found: C, 58.52; H, 3.43; N, 11.86.

PATENT

WO 2010085581

https://www.google.com/patents/WO2010085581A1?cl=en

Scheme 1

Scheme 2

Scheme 3

Scheme 4

Scheme 5

Scheme 6

Example 1

l-(4-(5-(3-Phenyl-4-(trifluoromethyl)isoxazol-5-yl)-l,2,4-oxadiazol-3- yl)benzyl)azetidine-3-carboxylic acid

1-A. 4,4,4-Trifluorobut-2-yn-l-ol

To a solution of diisopropylamine (24.7 mL, 176 mmol) in ether (100 mL) at -78 ⁰C was added a 1OM solution of butyllithium in ether (17.6 mL, 176 mmol) over 5 min. After 10 min. at -78°C, 2-bromo-3,3,3-trifluoroprop-l-ene (14.0 g, 80 mmol) was added to the pale yellow solution. After an additional 10 min., paraformaldehyde (2.40 g, 80 mmol) was added, the dry-ice bath was removed, and the reaction mixture was stirred at room temperature overnight. As the reaction mixture approached room temperature, it became dark in color. The reaction was quenched with a IN aqueous solution of hydrochloric acid (100 mL), diluted with ether (500 mL), washed with a IN aqueous solution of hydrochloric acid (2 x 100 mL), washed with brine 100 mL, and dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded a dark liquid which was distilled under low-vacuum (-50 Torr, ~50 ⁰C) to give 4,4,4-trifluorobut-2-yn-l-ol (7.1 g, 57.2 mmol, 72 % yield) as a pale yellow liquid. ¹H NMR (500 MHz, CDCl₃) δ ppm 2.31 (br. s., IH) and 4.38 – 4.42 (m, 2H).An Alternative Preparation of 1 -A: 4,4,4-Trifluorobut-2-yn- 1 -ol

-CF, (1-A) [00117] To an ether (pre-dried over magnesium sulfate) solution of phenanthroline (2.16 mg, 0.012 mmol) (indicator) at -78°C under nitrogen was added a 2M solution of n-butyl lithium in pentane. An orange color immediately appeared. Trifluoromethylacetylene gas was bubbled through the solution at -78°C. After ~4 min. of gas introduction, the orange color almost completely disappeared, the reaction solution became cloudy (due to some precipitation), and a pale light orange color persisted. Paraformaldehyde was added, and the dry ice/isopropanol bath was removed after 5 min. and replaced with a 0⁰C ice-bath. Stirring was continued for 45 min., the ice bath was removed, and stirring was continued for an additional 1.25 h. The reaction flask was immersed in a 0⁰C ice bath, and a saturated aqueous solution of ammonium chloride (20.0 mL) was added. The layers were separated, and the organic layer was washed with water (2x), washed with brine, and dried over anhydrous sodium sulfate. Concentration under low-vacuum (~50 Torr) without heat afforded a dark brown liquid which was purified by vacuum distillation (~50 Torr, -50 ⁰C) to give 4,4,4-trifluorobut-2-yn-l-ol (7.1 g, 57.2 mmol, 72 % yield) as a colorless liquid.

1-B. N-Hydroxybenzimidoyl chloride

This compound was prepared according to the method of Liu, K.C. et al, J. Org. Chem., 45:3916-1918 (1980).To a colorless, homogeneous solution of (E)-benzaldehyde oxime (24.4 g, 201 mmol) in N,N-dimethylformamide (60 mL) at room temperature was added N- chlorosuccinimide (26.9 g, 201 mmol) portion-wise over 30 min. During each addition, the reaction mixture would turn yellow and then gradually return to near colorlessness. Additionally, an exotherm was noted with each portion added to ensure that the reaction initiated after the addition of N-chlorosuccinimide. An ice bath was available, if required, to cool the exotherm. After the addition was complete, the homogeneous reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with 250 mL of water and extracted with ether (3 x 100 mL). The organic layers were combined, washed with water (2 x 100 mL), washed with a 10% aqueous solution of lithium chloride (2 x 100 mL), and washed with brine (100 mL). The aqueous layers were back extracted with ether (100 mL), and the combined organic layers (400 mL) were dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded (Z)-N-hydroxybenzimidoyl chloride (30.84 g, 198 mmol, 98 % yield) as a fluffy, pale yellow solid. The product had an HPLC ret. time = 1.57 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. LC/MS M⁺¹ = 155.8. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 7.30 – 7.64 (m, 3H), 7.73 – 7.87 (m, 2H), and 12.42 (s, IH).

l-C. 3-Phenyl-4-(trifluoromethyl)isoxazol-5-yl)methanol

To a pale yellow, homogeneous mixture of N-hydroxybenzimidoyl chloride (5.50 g, 35.4 mmol) and 4,4,4-trifluorobut-2-yn-l-ol (5.46 g, 39.6 mmol) in dichloroethane (85 mL) in a 250 mL round bottom flask at 70⁰C was added triethylamine (9.85 mL, 70.7 mmol) in 22 mL of dichloroethane over 2.5 h via an addition funnel (the first -50% over 2 h and the remaining 50% over 0.5 h). After the addition was complete, the reaction mixture was complete by HPLC (total time at 70⁰C was 3 h). The reaction mixture was stirred at room temperature overnight. [00121] The reaction mixture was diluted with dichloromethane (100 mL), washed with water (100 mL), and the organic layer was collected. The aqueous layer was extracted with dichloromethane (2 x 50 mL), and the combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. Analysis indicated that the product mixture was composed of a 86: 14 mixture of the desired regioisomer (1-C), (3-phenyl-4-(trifluoromethyl)isoxazol-5- yl)methanol, and the undesired regioisomer, (3-phenyl-5-(trifluoromethyl)isoxazol-4- yl)methanol. The mixture was purified by silica gel chromatography using a mixture of ethyl acetate and hexane (1% to pack and load – 5% – 9% – 12%) to afford (3- phenyl-4-(trifluoromethyl)isoxazol-5-yl)methanol (5.34 g, 21.96 mmol, 62.1 % yield) as a pale yellow oil. The compound had an HPLC ret. time = 1.91 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. LC/MS M⁺¹ =244.2. ¹H NMR (500 MHz, CDCl₃) δ ppm 2.21 (br. s., IH), 4.97 (s, 2H), 7.47 – 7.56 (m, 3H), and 7.65 (d, J=6.60 Hz, 2H).

1-D. 3-Phenyl-4-(trifluoromethyl)isoxazole-5-carboxylic acid

Preparation of Jones’ Reagent

To an orange, homogeneous solution of chromium trioxide (12.4 g, 0.123 mol) in water (88.4 mL) at 0⁰C was added sulfuric acid (10.8 mL) dropwise via addition funnel over 30 min. with stirring. The addition funnel was rinsed with water

(1 mL) to give 1.23 M solution of Jones’ Reagent (0.123 mol of reagent in 100 mL of solvent).

To a solution of (3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)methanol

(5.24 g, 21.6 mmol) in acetone (75 mL) at room temperature (immersed in a water bath) was added Jones’ Reagent (43.8 mL, 53.9 mmol) via addition funnel slowly over 1.5 h. The dark reaction mixture was stirred at room temperature overnight. By HPLC, the reaction was 93% complete. An additional 0.5 equivalents (9 mL) of the Jones’ Reagent was added. After 1 h, the reaction was 95% complete. After an additional 3h, the reaction was 96% complete. An additional 0.5 equivalents (9 mL) of the Jones’ Reagent was added. The reaction mixture was stirred for an additional 2.5 h. By HPLC, the reaction was 97% complete. Isopropyl alcohol (6 mL) was added, and the mixture was stirred for 90 min, resulting in a dark green precipitate. The mixture was diluted with ether (600 mL), washed with a 2% aqueous solution of sodium hydrogen sulfite (5 x 100 mL), and the organic layer was collected. The aqueous layer was back-extracted with ether (2 x 100 mL). By HPLC, there was no additional product in the aqueous layer. The combined organic layers were washed with water (100 mL), washed with a saturated aqueous solution of brine (100 mL), and dried over anhydrous sodium sulfate. The aqueous layer was back-extracted with ether (100 mL), and the organic layer was added to the previous organic layers. The solution was concentration under reduced pressure to give 3-phenyl-4-

(trifluoromethyl)isoxazole-5-carboxylic acid as an off-white solid. The solid was diluted with dichloromethane (200 mL), washed with a 2% aqueous solution of sodium hydrogen sulfite, washed with brine, and dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded 3-phenyl-4- (trifluoromethyl)isoxazole-5-carboxylic acid (3.84 g, 14.93 mmol, 69.3 % yield) as a pale yellow solid. The product was 96% pure by HPLC with a ret. time = 1.60 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. LC/MS M⁺¹ = 258.2. [00124] The sodium hydrogen sulfite aqueous layer still contained a significant amount of product. The brine layer contained no additional product and was discarded. The aqueous layer was saturated with sodium chloride, the pH was adjusted to -3.5, and the solution was extracted with ether (3 x 100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated to afford additional 3-phenyl-4-(trifluoromethyl)isoxazole-5-carboxylic acid (1.12 g, 4.36 mmol, 20.21 % yield) as a white solid. The product was >99% pure by HPLC with a ret. time = 1.60 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. LC/MS M⁺¹ = 258.1. ¹H NMR (500 MHz, DMSO-(I₆) δ ppm 7.55 – 7.63 (m, 5H). The products were combined to give 4.96 g (90% yield) of 3-phenyl-4- (trifluoromethyl)isoxazole-5-carboxylic acid.

An Alternative Preparation of 1-D: 3 -Phenyl -4-(trifluoromethyl)isoxazole-5- carboxylic acid starting with (3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)methanol

A mixture of (3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)methanol (2.1 g, 8.64 mmol), TEMPO (0.094 g, 0.604 mmol), and a sodium phosphate buffer (0.67M) (32.2 mL, 21.59 mmol) was heated to 35°C. A solution of sodium phosphate buffer (40 mL, pH -6.5) consisting of a 1: 1 solution OfNaH₂PO₄ (20 mL, 0.67M) and Na₂HPO₄ (20 mL, 0.67M) was prepared in acetonitrile (30 mL) was prepared prior to use. Solutions of sodium chlorite (3.91 g, 34.5 mmol) in water (4.5 mL) and bleach (4.3 mL, 6% wt.) were added simultaneously over 40min. The reaction was monitored by HPLC, and after 2 h, -30% of the starting material remained. After 6 h, 10% remained. Additional bleach (100 μL) was added, and the reaction mixture was left at room temperature overnight. [00127] Additional bleach (100 μL) was added. The resulting mixture was allowed to stir at 35°C for additional 2 h. HPLC indicated complete conversion. The reaction was quenched by the slow addition of a solution of sodium sulfite (2.07 mL, 43.2 mmol) in water (90 mL) at 0⁰C, resulting in the disappearance of the brown reaction color. The solvent was removed under reduced pressure, and the remaining aqueous residue was extracted with ethyl acetate (3 x 40 mL). The organic layers were combined, washed with water (8 mL), washed with brine (8 mL), and dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded 3 -phenyl – 4-(trifluoromethyl)isoxazole-5-carboxylic acid (2.2 g, 8.55 mmol, 99 % yield) as a pale yellow solid. An alternative procedure for the for the preparation of 3-phenyl-4-(trifluoromethyl) isoxazole-5-carboxylic acid starting with 4,4,4-trifluorobut-2ynoate (1-D)

Alt.1 -D- 1. Ethyl 3 -phenyl-4-(trifluoromethyl)isoxazole-5-carboxylate

To a pale yellow mixture of (Z)-N-hydroxybenzimidoyl chloride (1.04 g, 6.68 mmol) and ethyl 4,4,4-trifluorobut-2-ynoate (1.238 g, 7.45 mmol) in diethyl ether (20 mL) at room temperature was added triethylamine (1.86 mL, 13.4 mmol) over 15 min., resulting in a precipitant. After the addition was complete, the pale yellow slurry was stirred at room temperature over a weekend. The heterogeneous reaction mixture was filtered under reduced pressure to remove the triethylamine hydrochloride salt, and the filtrate was concentrated to give the product mixture as a dark yellow, viscous oil (2.03 g). By HPLC, the reaction mixture was composed of a mixture of the desired regioisomer, ethyl 3-phenyl-4-(trifluoromethyl)isoxazole-5- carboxylate, and the undesired regioisomer, ethyl 3-phenyl-5- (trifluoromethyl)isoxazole-4-carboxylate, in an approximately 15:85 ratio. The compound mixture was dissolved in hexane and sonicated for 5 min. The hexane was decanted off, and the dark red, oily residue was found to have only trace product by HPLC. The hexane was removed under reduced pressure, and the residue (1.89 g) was purified by preparative HPLC. The desired fractions containing ethyl 3-phenyl- 4-(trifluoromethyl)isoxazole-5-carboxylate were concentrated, and the residue was diluted with dichloromethane, washed with a saturated aqueous solution of sodium bicarbonate, and dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded ethyl 3-phenyl-4-(trifluoromethyl) isoxazole-5-carboxylate (0.087 g, 0.305 mmol, 4.6 % yield) as a pale yellow solid. The compound had an HPLC ret. time = 2.88 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.46 (t, J=7.15 Hz, 3H), 4.53 (q, J=7.03 Hz, 2H), 7.48 – 7.55 (m, 3H), and 7.58 (d, J=7.53 Hz, 2H).

An Alternative Preparation of 1-D-l : Ethyl 3-phenyl-4-(trifluoromethyl)isoxazole-5- carboxylic acid starting with ethyl 4,4,4-trifluorobut-2-enoate

1-D-l. Ethyl 2,3-dibromo-4,4,4-trifluorobutanoate

Br L _/COOEt

Br (1-D-l) [00129] Bromine (18.4 mL, 357 mmol) was added dropwise over 30 minutes to a solution of (E)-ethyl 4,4,4-trifluorobut-2-enoate (50 g, 297 mmol) in carbon tetrachloride (50 mL) at room temperature under nitrogen. The resulting dark red solution was refluxed for 4 hours. Additional bromine (2ml) was added and heating was continued until the HPLC analysis showed that the starting material had been consumed. The reaction mixture was concentrated under reduced pressure to give light brown oil which used in the next step without purification. HPLC (XBridge 5μ Cl 8 4.6×50 mm, 4 mL/min, Solvent A: 10 % MeOH/water with 0.2 % H₃PO₄, Solvent B: 90 % MeOH/water with 0.2 % H₃PO₄, gradient with 0-100 % B over 4 minutes): 2.96 and 3.19 minutes.

l-D-2. (Z/E)-Ethyl 2-bromo-4,4,4-trifluorobut-2-enoate

,COOEt

F₃C

Br (l-D-2)

To a solution of ethyl 2,3-dibromo-4,4,4-trifluorobutanoate (1-B-l) in hexane (200 mL) cooled to 0⁰C was added triethylamine (49.7 ml, 357mmol) drop- wise over 35 minutes, during which time a white precipitate formed. The reaction mixture was stirred for an additional 2 hours until LC indicated complete conversion. The solid was filtered and rinsed with hexane (3 x 5OmL), and the filtrate was concentrated and passed through a short silica gel pad eluting with 10% ethyl acetate/hexane to give (Z/E)-ethyl 2-bromo-4,4,4-trifluorobut-2-enoate (65.5 g, 265mmol, 89 % yield for two steps) as a colorless oil. Alternatively, the crude product can be purified by distillation (85 ⁰C / -60 mmHg). ¹H NMR (CDCl₃, 400 MHz) 5 7.41 (q, IH, J= 7.28 Hz), 4.35 (q, 2H, J= 7.11 Hz), 1.38 (t, 3H, J= 7.15 Hz); HPLC (XBridge 5μ Cl 8 4.6×50 mm, 4 mL/min, Solvent A: 10 % MeOH/water with 0.2 % H₃PO₄, Solvent B: 90 % MeOH/water with 0.2 % H₃PO₄, gradient with 0- 100 % B over 4 minutes): 3.09 minutes.

1-D-l. Ethyl 3 -phenyl -4-(trifluoromethyl)isoxazole-5-carboxylate

(Z/E)-Ethyl 2-bromo-4,4,4-trifluorobut-2-enoate, l-D-3, (39.7 g, 161 mmol) and N-hydroxybenzimidoyl chloride (30 g, 193mmol) were dissolved in ethyl acetate (15OmL). Indium (III) chloride (8.89 g, 40.2mmol) was added and the resulting mixture stirred for 60 minutes at RT under N₂. Potassium hydrogen carbonate (32.2 g, 321mmol) was added to the reaction mixture which was allowed to stir overnight for 14 hours at RT. The solvent was removed in vacuo. The residue was re-suspended in 30OmL hexane and stirred for lOmiutes then filtered. The filter cake was washed with hexane (3X3 OmL) and the combined filtrate was concentrated in vacuo to give crude product, which was further purified with flash chromatography to generate 33g product (72%) as light yellowish oil as a mixture of the desired isomer 1-D-l and undesired isomer 1-D-la in a ratio of -30/1. MS m/e 286.06(M+H⁺); ¹H NMR (CDCl₃, 400 MHz) δ 7.56 (m, 5H), 4.53 (q, 2H, J= 7.3 Hz), 1.46 (t, 3H, J= 7.2 Hz); HPLC (XBridge 5μ C18 4.6×50 mm, 4 mL/min, Solvent A: 10 % MeOH/water with 0.2 % H₃PO₄, Solvent B: 90 % MeOH/water with 0.2 % H₃PO₄, gradient with 0-100 % B over 4 minutes): 3.57 minutes.

Alt.1-D. 3-Phenyl-4-(trifluoromethyl)isoxazole-5-carboxylic acid, lithium salt

A mixture of ethyl 3-phenyl-4-(trifluoromethyl)isoxazole-5-carboxylate, 1-D-l, (0.085 g, 0.298 mmol) and lithium hydroxide hydrate (0.013 g, 0.298 mmol) in methanol (2.0 mL) and water (1.0 mL) was stirred at room temperature overnight. The reaction mixture was concentrated to dryness to give 3-phenyl-4- (trifluoromethyl)isoxazole-5-carboxylic acid, lithium salt (0.079 g, 0.299 mmol, 100 % yield) as a pale yellow solid. The compound had an HPLC ret. time = 1.72 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. LC/MS M⁺¹ = 258.0. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.49 – 7.57 (m, 3H) and 7.58 – 7.62 (m, 2H).1-E. 3-Phenyl-4-(trifluoromethyl)isoxazole-5-carbonyl fluoride

To a mixture of 3-phenyl-4-(trifluoromethyl)isoxazole-5-carboxylic acid (3.00 g, 11.7 mmol) and pyridine (1.132 mL, 14.0 mmol) in dichloromethane (100 mL) at room temperature was added 2,4,6-trifluoro-l,3,5-triazine (cyanuric fluoride) (1.18 mL, 14.0 mmol). The reaction mixture was stirred at room temperature overnight, diluted with dichloromethane (300 mL), washed with an ice-cold solution of 0.5N aqueous hydrochloric acid (2 x 100 mL), and the organic layer was collected. The aqueous layer was back-extracted with dichloromethane (200 mL), and the combined organic layers were dried anhydrous sodium sulfate and concentrated to afford 3-phenyl-4-(trifluoromethyl)isoxazole-5-carbonyl fluoride (2.91 g, 11.2 mmol, 96 % yield) as a yellow, viscous oil. The product was found to react readily with methanol and on analysis was characterized as the methyl ester, which had an HPLC ret. time = 2.56 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. LC/MS M⁺¹ = 272.3 (methyl ester).1-F. tert-Butyl l-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)-l,2,4-oxadiazol- 3-yl)-benzyl)azetidine-3-carboxylate

A suspension of 3-phenyl-4-(trifluoromethyl)isoxazole-5-carbonyl fluoride (2.91 g, 11.2 mmol), (Z)-tert-butyl 1-(4-(N’- hydroxycarbamimidoyl)benzyl)azetidine-3-carboxylate (Int. l, 3.43 g, 11.2 mmol), and Hunig’s Base (2.55 mL, 14.6 mmol) in acetonitrile (20 mL) was stirred at room temperature over the weekend. The reaction mixture had completely solidified (pinkish-tan in color), but was judged complete by HPLC and LCMS. The reaction mixture was partitioned between a saturated aqueous of sodium bicarbonate (150 mL) and dichloromethane (150 mL). The aqueous layer was extracted with dichloromethane (2 x 100 mL), and the combined organic layers were dried over anhydrous sodium sulfate. Concentration under reduced pressure afforded a tan solid which was purified by flash silica gel chromatography using a mixture of ethyl acetate in hexane (0-50%) to afford tert-butyl l-(4-(5-(3-phenyl-4-(trifluoromethyl) isoxazol-5-yl)-l,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (4.60 g; 78%) as a white, crystalline solid. The material was suspended in methanol (-75 mL) and was sonicated for 5 minutes. The MeOH was removed under reduce pressure, and the residue was re-suspended in methanol (-50 mL) with sonication. Vacuum filtration and drying afforded tert-butyl l-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)- l,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (4.04 g, 7.67 mmol, 68 % yield) as a white, crystalline solid. The methanol filtrate was concentrated to afford additional tert-butyl l-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)- 1,2,4- oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (570 mg; 10%) as a slightly off- white solid. The compound had an HPLC retention time = 3.12 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. LC/MS M⁺¹ =527.1. ¹H NMR (500 MHz, CDCl₃) δ ppm 1.47 (s, 9H) 3.28 – 3.37 (m, 3H), 3.60 (br. s., 2H), 3.74 (br. s., 2H), 7.49 (d, J=7.70 Hz, 2H), 7.53 – 7.62 (m, 3H), 7.69 (d, J=7.15 Hz, 2H), and 8.16 (d, J=7.70 Hz, 2H). 1. Preparation of l-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5-yl)-l,2,4- oxadiazol-3-yl)benzyl)azetidine-3-carboxylic acid

A mixture of tert-butyl l-(4-(5-(3-phenyl-4-(trifluoromethyl)isoxazol-5- yl)-l,2,4-oxadiazol-3-yl)benzyl)azetidine-3-carboxylate (6.12 g, 11.6 mmol) and trifluoroacetic acid (50.1 mL, 651 mmol) was stirred at room temperature for 1.5 h. By HPLC, the deprotection appeared to be complete after 1 h. The TFA was removed under reduced pressure, and the oily residue was diluted with water (100 mL) and sonicated for 5 min. The resulting suspension was stirred for an additional 10 min until a consistent white suspension was observed. A IN aqueous solution of sodium hydroxide was added portion-wise until the pH was ~4.5 (42 mL of IN NaOH). Over time, the pH drifted back down to 3-4, and additional IN aqueous sodium hydroxide had to be added. The suspension was stirred overnight at room temperature. Several drops of IN aqueous sodium hydroxide were added to re-adjust the pH to 4.5, and after 60 min., the pH appeared to be stable. The solid was collected by vacuum filtration, washed with water several times, and dried under reduced pressure for 5 h. The solid was then suspended in methanol (110 mL) in a 150 mL round bottom flask and sonicated for 15 min. During the sonication, the solution became very thick. An additional 25 mL of methanol was added, and the suspension was stirred overnight. The product was collected by vacuum filtration, washed with methanol (-50 mL), and dried under reduced pressure. The solid was transferred to a 250 mL round bottom flask, re-suspended in methanol (115 mL), sonicated for 5 min., and stirred for 60 min. The solid was collected by vacuum filtration, washed with methanol (~50 mL), and dried over well under reduced pressure to give l-(4-(5-(3- phenyl-4-(trifluoromethyl)isoxazol-5-yl)-l,2,4-oxadiazol-3-yl)benzyl)azetidine-3- carboxylic acid (5.06 g, 10.7 mmol, 92 % yield) as a crystalline, white solid. The product had an HPLC ret. time = 2.79 min. – Column: CHROMOLITH® SpeedROD 4.6 x 50 mm (4 min.); Solvent A = 10% MeOH, 90% H₂O, 0.1% TFA; Solvent B = 90% MeOH, 10% H₂O, 0.1% TFA. LC/MS M⁺¹ = 471.3. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.20 – 3.46 (m, 5H), 3.66 (s, 2H), 7.53 (d, J=8.25 Hz, 2H), 7.60 – 7.70 (m, 5H), and 8.06 (d, J=7.70 Hz, 2H).

HPLC purity 100/99.8%, ret. time = 7.62 min. (A linear gradient using 5% acetonitrile, 95% water, and 0.05% TFA (Solvent A) and 95% acetonitrile, 5% water, and 0.05% TFA (Solvent B); t = 0 min., 10% B, t = l2 min., 100% B (15 min.) was employed on a SunFire C18 3.5u 4.6 x 150 mm column. Flow rate was 2 ml/min and UV detection was set to 220/254 nm.).

HPLC purity 100/99.9%, ret. time = 8.45 min. (A linear gradient using 5% acetonitrile, 95% water, and 0.05% TFA (Solvent A) and 95% acetonitrile, 5% water, and 0.05% TFA (Solvent B); t = 0 min., 10% B, t = l2 min., 100% B (15 min.) was employed on a XBridge Ph 3.5u 4.6 x 150 mm column. Flow rate was 2 ml/min and UV detection was set to 220/254 nm.).

CONSTRUCTION

Alt.1 -D- 1. Ethyl 3 -phenyl-4-(trifluoromethyl)isoxazole-5-carboxylate

ADDITIONAL INFORMATION

Sphingosine 1-phosphate (S1P) is the endogenous ligand for the sphingosine 1-phosphate receptors (S1P1–5) and evokes a variety of cellular responses through their stimulation. The interaction of S1P with the S1P receptors plays a fundamental physiological role in a number of processes including vascular development and stabilization, lymphocyte migration, and proliferation

REFERENCES

Watterson, S. H.; Guo, J.; Spergel, S. H.; Langevine, C. L.; Moquin, R. V.; Shen, D.
R.; Yarde, M.; Cvijic, M. E.; Banas, D.; Liu, R.; Suchard, S. J.; Gillooly, K.; Taylor,
T.; Rex-Rabe, S.; Shuster, D. J.; McIntyre, K. W.; Cornelius, G.; Darienzo, C.;
Marino, A.; Balimane, P.; Warrack, B.; Saltercid, L.; McKinnon, M.; Barrish, J. C.;
Carter, P. C.; Pitts, W. J.; Xie, J.; Dyckman, D. J. J. Med. Chem. 2016, 59, 2820.

Watterson, S.H.; Guo, J.; Spergel, S.H.; et al.
Potent and selective agonists of Sphingosine-1-Phosphate 1 (S1P1): The discovery and SAR of a novel isoxazole based series
241st Am Chem Soc (ACS) Natl Meet (March 27-30, Anaheim) 2011, Abst MEDI 96

/////Potent and Selective Isoxazole-Containing S1P₁ Receptor Agonist, BMS 520, Sphingosine-1-Phosphate 1 (S1P1)

O=C(C1CN(CC2=CC=C(C3=NOC(C4=C(C(F)(F)F)C(C5=CC=CC=C5)=NO4)=N3)C=C2)C1)O

A New Antibiotic (E)-3-(3-Carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one, from University Of Notre Dame

PRECLINICAL, Uncategorized Comments Off

May 062016

(E)-3-(3-Carboxyphenyl)-2-(4-ethynylstyryl)quinazolin-4(3H)-one

(E)-3-(2-(4-Cyanostyryl)-4-Oxoquinazolin-3(4h)-Yl)benzoic Acid;

1624273-22-8 CAS

NA SALT 1624273-21-7 CAS

INNOVATORS

University Of Notre Dame

Mayland Chang, Shahriar Mobashery, Renee BOULEY INVENTORS

C₂₄H₁₅N₃O₃
Molecular Weight:	393.3942 g/mol

¹H NMR (500 MHz, DMSO-d₆) δ 4.32 (s, 1H), 6.34 (d, J = 15.55 Hz, 1H), 7.35 (d, J = 8.37 Hz, 2H), 7.44 (d, J = 8.37 Hz, 2H), 7.49 (d, J = 7.58 Hz, 1H), 7.55 (t, J = 7.98 Hz, 1H), 7.58 (t, J = 7.78 Hz, 1H), 7.78 (d, J = 8.17 Hz, 1H), 7.87 (m, 3H), 8.05 (d, J = 7.78 Hz, 1H), 8.13 (d, J = 7.98 Hz, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 82.70, 83.24, 120.66, 121.04, 122.84, 126.51, 126.81, 127.31, 127.83, 129.98, 130.12, 132.33, 132.39, 133.49, 134.90, 135.21, 137.21, 137.99, 147.36, 151.04, 161.37, 166.58.

HRMS (m/z): [M + H]⁺, calcd for C₂₅H₁₇N₂O₃, 393.1234; found, 393.1250. HRMS (m/z): [M + Na]⁺, calcd for C₂₅H₁₆N₂NaO₃, 415.1053; found, 415.1054.

The emergence of resistance to antibiotics over the past few decades has created a state of crisis in the treatment of bacterial infections.Over the years, β-lactams were the antibiotics of choice for treatment of S. aureus infections. However, these agents faced obsolescence with the emergence of methicillin-resistant S. aureus (MRSA). Presently, vancomycin, daptomycin, linezolid, or ceftaroline are used for treatment of MRSA infections, although only linezolid can be dosed orally. Resistance to all four has emerged. Thus, new anti-MRSA antibiotics are sought, especially agents that are orally bioavailable. a new antibiotic (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)–one, with potent activity against S. aureus, including MRSA. We document that quinazolinones of our design are inhibitors of cell-wall biosynthesis in S. aureus and do so by binding to dd-transpeptidases involved in cross-linking of the cell wall. quinazolinones possess activity in vivo and are orally bioavailable. This antibiotic holds promise in treating difficult infections by MRSA.

PAPER

Journal of the American Chemical Society (2015), 137(5), 1738-1741.

http://pubs.acs.org/doi/abs/10.1021/jacs.5b00056

Discovery of Antibiotic (E)-3-(3-Carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one

Renee Bouley^†, Malika Kumarasiri^†, Zhihong Peng^†, Lisandro H. Otero^‡, Wei Song^†, Mark A. Suckow^§, Valerie A. Schroeder^§, William R. Wolter^§, Elena Lastochkin^†, Nuno T. Antunes^†, Hualiang Pi^†, Sergei Vakulenko^†, Juan A. Hermoso^‡, Mayland Chang^*^†, and Shahriar Mobashery^*^†

^† Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States

^‡ Department of Crystallography and Structural Biology, Instituto de Química-Física “Rocasolano”, Consejo Superior de Investigaciones Científicas, Madrid, Spain

^§ Freimann Life Sciences Center and Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, United States

J. Am. Chem. Soc., 2015, 137 (5), pp 1738–1741

DOI: 10.1021/jacs.5b00056

Publication Date (Web): January 28, 2015

*mchang@nd.edu, *mobashery@nd.edu

Abstract

In the face of the clinical challenge posed by resistant bacteria, the present needs for novel classes of antibiotics are genuine. In silico docking and screening, followed by chemical synthesis of a library of quinazolinones, led to the discovery of (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)–one (compound 2) as an antibiotic effective in vivo against methicillin-resistant Staphylococcus aureus (MRSA). This antibiotic impairs cell-wall biosynthesis as documented by functional assays, showing binding of 2 to penicillin-binding protein (PBP) 2a. We document that the antibiotic also inhibits PBP1 of S. aureus, indicating a broad targeting of structurally similar PBPs by this antibiotic. This class of antibiotics holds promise in fighting MRSA infections.

PATENT

WO 2014138302

http://www.google.com/patents/WO2014138302A1?cl=en

Staphylococcus aureus is a common bacterium found in moist areas of the body and skin. S. aureus can also grow as a biofilm, representing the leading cause of infection after implantation of medical devices. Approximately 29% (78.9 million) of the US population is colonized in the nose with S. aureus, of which 1.5% (4.1 million) is methicillin-resistant S. aureus (MRSA). In 2005, 478,000 people in the US were hospitalized with a S. aureus infection, of these 278,000 were MRSA infections, resulting in 19,000 deaths. MRSA infections have been increasing from 2% of S. aureus infections in intensive care units in 1974 to 64% in 2004, although more recent data report stabilization. Approximately 14 million outpatient visits occur every year in the US for suspected S. aureus skin and soft tissue infections. About 76% of these infections are caused by S. aureus, of which 78% are due to MRSA, for an overall rate of 59%. Spread of MRSA is not limited to nosocomial (hospital-acquired) infections, as they are also found in community-acquired infections. Over the years, β-lactams were antibiotics of choice in treatment of S. aureus infections. However, these agents faced obsolescence with the emergence of

MRSA. Presently, vancomycin, daptomycin or linezolid are agents for treatment of MRSA infections, although only linezolid can be dosed orally. Resistance to all three has emerged. Thus, new anti-MRSA therapeutic strategies are needed, especially agents that are orally bioavailable.

Clinical resistance to β-lactam antibiotics by MRSA has its basis predominantly in acquisition of the mecA gene, which encodes penicillin-binding protein 2a (PBP2a). PBP2a, a cell-wall DD- transpeptidase, is refractory to inhibition by essentially all commercially available β-lactams (ceftaroline is an exception), antibiotics that irreversibly acylate the active-site serine of typical PBPs. PBPs catalyze biosynthesis of the bacterial cell wall, which is essential for the survival of the bacterium. Accordingly, new ηοη-β-lactam antibiotics that inhibit PBP2a are needed to combat drug-resistant strains of bacteria. SUMMARY

Staphylococcus aureus is responsible for a number of human diseases, including skin and soft tissue infections. Annually, 292,000 hospitalizations in the US are due to S. aureus infections, of which 126,000 are related to methicillin-resistant Staphylococcus aureus (MRSA), resulting in 19,000 deaths. A novel structural class of antibiotics has been discovered and is described herein. A lead compound in this class shows high in vitro potency against Gram-positive bacteria comparable to those of linezolid and superior to vancomycin (both considered gold standards) and shows excellent in vivo activity in mouse models of MRSA infection.

The invention thus provides a novel class of ηοη-β-lactam antibiotics, the quinazolinones, which inhibit PBP2a by an unprecedented mechanism of targeting both its allosteric and active sites. This inhibition leads to the impairment of the formation of cell wall in living bacteria. The quinazolinones described herein are effective as anti-MRSA agents both in vitro and in vivo. Furthermore, they exhibit activity against other Gram-positive bacteria. The quinazolinones have anti-MRSA activity by themselves. However, these compounds synergize with β-lactam antibiotics. The use of a combination of a quinazolinone with a β-lactam antibiotic can revive the clinical use of β-lactam antibacterial therapy in treatment of MRSA infections. The invention provides a new class of quinazolinone antibiotics, optionally in combination with other antibacterial agents, for the therapeutic treatment of methicillin- resistant Staphylococcus aureus and other bacteria.

The quinazolinone compounds described herein can be prepared using standard synthetic techniques known to those of skill in the art. Examples of such techniques are described by Khajavi et al. (J. Chem. Res. (S), 1997, 286-287) and Mosley et al. (J. Med. Chem. 2010, 53, 5476-5490). A general preparatory scheme for preparing the compounds described herein, for example, compounds of Formula

wherein each of the variables are as defined for one or more of the formulas described herein, such as Formula (A).

EXAMPLES

Example 1. Compound Preparation

Chemistry. Organic reagents and solvents were purchased from Sigma- Aldrich. ^lH and ¹³C NMR spectra were recorded on a Varian INOVA-500. High-resolution mass spectra were obtained using a Bruker micrOTOF/Q2 mass spectrometer.

2-Methyl-4H-benzo[</| [l,3]oxazin-4-one (3). Anthranilic acid (20 g, 146 mmol) was dissolved in triethyl orthoacetate (45 mL, 245 mmol) and refluxed for 2 h. The reaction mixture was cooled on ice for 4 h to crystallize the intermediate. The resulting crystals were filtered and washed with hexanes to give 3 (17 g, 72% yield). ^lH NMR (500 MHz, CDC1₃) δ 2.47 (s, 3H), 7.50 (t, J= 7.38 Hz, 1H), 7.54 (d, J = 7.98 Hz, 1H), 7.80 (t, J= 7.18 Hz, 1H), 8.18 (d, J= 7.78 Hz, 1H). ¹³C NMR (126 MHz, CDCI3) δ 21.59, 1 16.84, 126.59, 128.42, 128.66, 136.77, 146.61, 159.89, 160.45. HRMS (m/z): [M + H]⁺, calcd for C9H8NO2, 162.0550; found , 162.0555.

2-Methyl-3-(3-carboxyphenyl)-quinazolin-4(3//)-one (4). Compound 3 (2 g, 12.4 mmol) and 3- aminophenol (1.7 g, 12.4 mmol) were suspended in glacial acetic acid (8 mL, 140 mmol), and dissolved upon heating. The reaction was refluxed for 5 h, at which point 5 mL water was added to the cooled reaction mixture. The resulting precipitate was filtered and washed with water, followed by cold ethanol and hexane to give 4 (3.19 g, 92% yield). ^lH (500 MHz, DMSO-d₆) δ 2.87 (s, 3H), 7.52 (t, J= 7.38 Hz, 1H), 7.66-7.73 (m, 3H), 7.84 (t, J= 7.38 Hz, 1H), 8.01 (s, 1H), 8.09 (t, J= 7.58 Hz, 2H). ¹³C NMR (126 MHz, DMSO-de) δ 24.13, 120.48, 126.32, 126.47, 126.72, 129.52, 129.83, 130.01, 132.40, 133.07, 134.67, 138.18, 147.37, 154.13, 161.44, 166.58. HRMS (m/z): [M + H]⁺, calcd for C16H13N2O3 ,

281.0921 ; found, 281.0917.

Sodium (£^‘)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one (2). Compound 4 (1.0 g, 3.6 mmol) and 4-formylbenzonitrile (0.56 g, 4.3 mmol) were suspended in glacial acetic acid (5 mL, 87 mmol), a suspension that dissolved upon heating. The reaction was refluxed for 18 h and 5 mL water was added to the cooled reaction mixture. The resulting precipitate was filtered and washed with water, followed by cold ethanol and hexanes to afford the carboxylic acid (0.77g, 75% yield). HRMS (m/z): [M + H]⁺, calcd for C24H16N3O3, 394.1 186; found 394.1214. The carboxylic acid (0.45 g, 1.1 mmol) was dissolved in hot ethanol, to which sodium 2-ethylhexanoate (0.28 g, 1.7 mmol) was added. The reaction mixture was stirred on ice for 2 h. The precipitate was filtered and washed with cold ethanol. The product was obtained by dissolving the precipitate in about 5 mL of water and subsequent lyophilization of the solution to give 2 as the sodium salt (0.4 g, 85% yield).

¾ NMR (500 MHz, DMSO- de) δ 6.47 (d, J= 15.55 Hz, 1H), 7.59 (m, 3H), 7.74 (d, J= 5.38 Hz, 2H), 7.79 (m, 3H), 7.91 (m, 2H), 8.05 (s, 1H), 8.14 (d, J= 7.78 Hz, 2H).

¹³C NMR (126 MHz, DMSO-de) δ 11 1.56, 1 18.61, 120.76, 123.42, 126.50, 127.01, 127.35, 128.26, 129.99, 130.06, 130.12, 132.33, 132.83, 133.46, 134.89, 136.95, 137.03, 139.25, 147.21, 150.74, 161.25, 166.52.

HRMS (m/z): [M + H]⁺, calcd for C24Hi₅N₃NaO₃, 416.1006; found, 416.0987.

PAPER

http://pubs.acs.org/doi/full/10.1021/acs.jmedchem.6b00372

Structure–Activity Relationship for the 4(3H)-Quinazolinone Antibacterials

Renee Bouley^†, Derong Ding^†, Zhihong Peng^†, Maria Bastian^†, Elena Lastochkin^†, Wei Song^†, Mark A. Suckow^‡,Valerie A. Schroeder^‡, William R. Wolter^‡, Shahriar Mobashery^*^†, and Mayland Chang^*^†

^† Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States

^‡ Freimann Life Sciences Center and Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, United States

J. Med. Chem., Article ASAP

DOI: 10.1021/acs.jmedchem.6b00372

Publication Date (Web): April 18, 2016

*S.M.: e-mail, mobashery@nd.edu; phone, 574-631-2933., *M.C.: e-mail, mchang@nd.edu; phone, 574-631-2965.

ACS Editors’ Choice – This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

We recently reported on the discovery of a novel antibacterial (2) with a 4(3H)-quinazolinone core. This discovery was made by in silico screening of 1.2 million compounds for binding to a penicillin-binding protein and the subsequent demonstration of antibacterial activity againstStaphylococcus aureus. The first structure–activity relationship for this antibacterial scaffold is explored in this report with evaluation of 77 variants of the structural class. Eleven promising compounds were further evaluated for in vitro toxicity, pharmacokinetics, and efficacy in a mouse peritonitis model of infection, which led to the discovery of compound 27. This new quinazolinone has potent activity against methicillin-resistant (MRSA) strains, low clearance, oral bioavailability and shows efficacy in a mouse neutropenic thigh infection model.

NMR

INVENTORS

Renee Bouley

Renee Bouley selected to receive prestigious ACS Predoctoral Fellowship

Published: July 02, 2013

Renee Bouley

Renee Bouley, a third year graduate student in the Department of Chemistry and Biochemistry, has been selected to receive a prestigious American Chemical Society (ACS) Division of Medicinal Chemistry Predoctoral Fellowship. Bouley is one of only four recipients chosen for the 2013-2014 cycle.

This award supports doctoral candidates working in the area of medicinal chemistry who have demonstrated superior achievements as graduate students and who show potential for future work as independent investigators. These fellowships have been awarded annually since 1991 and include one year stipend support and an invitation to present the fellow’s research results at a special awards session at the ACS National Meeting.

Bouley’s work, conducted under the advisement of Shahriar Mobashery, Navari Family Professor in Life Sciences, and Mayland Chang, Research Professor and Director of the Chemistry-Biochemistry-Biology Interface (CBBI) Program, centers around the discovery of a new class of antibiotics that are selective against staphylococcal species of bacteria, including hard-to-treat methicillin-resistant Staphylococcus aureus (MRSA). She has already identified a class of compounds that has in vitro activity against bacteria and demonstrated efficacy in mice. Bouley spent three months in 2012 in the laboratory of Prof. Juan Hermoso at Consejo Superior de Investigaciones Cientificas in Madrid, Spain, where she solved the crystal structure of the lead compound in complex with its target protein. Her studies have shown an unprecedented mechanism of action that opens opportunities for clinical resurrection of β-lactam antibiotics in combination with the new antibiotics. Bouley’s work during her fellowship tenure will explore structural analogs of these compounds with the goal of optimizing their potency in vivo and improving their drug-like properties.

Bouley is already the recipient of a National Institutes of Health Ruth L. Kirschstein National Research Service Award – CBBI (Chemistry-Biochemistry-Biology Interface) Program, a CBBI Research Internship Award, and an American Heart Association Predoctoral Fellowship (declined)………..https://www.linkedin.com/in/renee-bouley-43243215

University of Notre Dame

MAYLAND CHANG

http://chemistry.nd.edu/people/mayland-chang/

MAYLAND CHANG

Research Professor; Director, Chemistry-Biochemistry-Biology Interface (CBBI) Program
Office: 247 NSH
Phone: (574) 631-2965

Dr. Chang obtained B.S. degrees in biological sciences and chemistry from the University of Southern California, and a Ph.D. in chemistry from the University of Chicago. Subsequently, she conducted postdoctoral research at Columbia University as a National Institutes of Health postdoctoral fellow. She joined the faculty of the University of Notre Dame in 2003. Previously, Dr. Chang was Chief Operating Officer of University Research Network, Inc., Senior Scientist with Pharmacia Corporation, and Senior Chemist at Dow Chemical Company. She has characterized the ADME properties of numerous drugs, as well as prepared NDAs, INDs, Investigator’s Brochures, product development plans, and candidate drug evaluations.

Shahriar Mobashery

Navari Professor at University of Notre Dame

The Mobashery research program integrates computation, biochemistry, molecular biology, and the organic synthesis of medically important molecules. Bringing together these different disciplines is required to produce both scientific and medical advances for very difficult, but critically important clinical problems.

http://chemistry.nd.edu/people/shahriar-mobashery/

https://www.linkedin.com/in/shahriar-mobashery-71b67b4b

/////// 1624273-22-8, Antibiotic, (E)-3-(3-Carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one, methicillin-resistant S. aureus, MRSA, 1624273-21-7, PRECLINICAL

O=C(O)c1cc(ccc1)N3C(=Nc2ccccc2C3=O)/C=C/c4ccc(C#N)cc4

VX-? , an Azaindolyl-Pyrimidine Inhibitor of Influenza Virus Replication from Vertex

PRECLINICAL, Uncategorized Comments Off

Apr 302016

VX-?

An Azaindolyl-Pyrimidine Inhibitor of Influenza Virus Replication from Vertex

SYNTHESIS COMING……..

CAS 1259498-06-0

MF C₂₃ H₂₇ F₂ N₇ O, MW, 455.50

1-Piperidinecarboxamide, N-[(1R,3S)-3-[[5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-pyrimidinyl]amino]cyclohexyl]-

N-[(1R,3S)-3-[[5-Fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl]amino]cyclohexyl]morpholine-4-carboxamide

N-[(1R,3S)-3-[[5-Fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl]amino]cyclohexyl]morpholine-4-carboxamide , (1R,3S)-cis-diaminocyclohexane.

Specific Rotation

[α]²¹_D = −165.7° (c = 1 in MeOH).

¹H NMR (300 MHz, d₆-DMSO) δ 12.23 (s, 1H), 8.42 (dd, J = 9.8, 2.9 Hz, 1H), 8.34–8.18 (m, 2H), 8.14 (d, J = 4.0 Hz, 1H), 7.49 (d, J = 7.5 Hz, 1H), 6.33 (d, J= 7.6 Hz, 1H), 4.24–4.00 (m, 1H), 3.75–3.57 (m, 1H), 3.57–3.42 (m, 4H), 3.28–3.09 (m, 4H), 2.15 (d, J = 11.4 Hz, 1H), 2.01 (d, J = 11.2 Hz, 1H), 1.83 (d, J = 9.7 Hz, 2H), 1.60–1.07 (m, 4H).¹⁹F NMR (282.4 MHz, d₆-DMSO) −138.10, −158.25 ppm.

HRMS (ESI) [M + H]⁺ calculated for C₂₂H₂₆F₂N₇O₂ 458.2111, found 458.2110.

Influenza spreads around the world in seasonal epidemics, resulting in the deaths of hundreds of thousands annually – millions in pandemic years. For example, three influenza pandemics occurred in the 20th century and killed tens of millions of people, with each of these pandemics being caused by the appearance of a new strain of the virus in humans. Often, these new strains result from the spread of an existing influenza virus to humans from other animal species.

Influenza is primarily transmitted from person to person via large virus-laden droplets that are generated when infected persons cough or sneeze; these large droplets can then settle on the mucosal surfaces of the upper respiratory tracts of susceptible individuals who are near (e.g. within about 6 feet) infected persons. Transmission might also occur through direct contact or indirect contact with respiratory secretions, such as touching surfaces contaminated with influenza virus and then touching the eyes, nose or mouth. Adults might be able to spread influenza to others from 1 day before getting symptoms to approximately 5 days after symptoms start. Young children and persons with weakened immune systems might be infectious for 10 or more days after onset of symptoms. [00103] Influenza viruses are RNA viruses of the family Orthomyxoviridae, which comprises five genera: Influenza virus A, Influenza virus B, Influenza virus C, Isavirus and Thogoto virus.

The Influenza virus A genus has one species, influenza A virus. Wild aquatic birds are the natural hosts for a large variety of influenza A. Occasionally, viruses are transmitted to other species and may then cause devastating outbreaks in domestic poultry or give rise to human influenza pandemics. The type A viruses are the most virulent human pathogens among the three influenza types and cause the most severe disease. The influenza A virus can be subdivided into different serotypes based on the antibody response to these viruses. The serotypes that have been confirmed in humans, ordered by the number of known human pandemic deaths, are: HlNl (which caused Spanish influenza in 1918), H2N2 (which caused Asian Influenza in 1957), H3N2 (which caused Hong Kong Flu in 1968), H5N1 (a pandemic threat in the 2007-08 influenza season), H7N7 (which has unusual zoonotic potential), H1N2 (endemic in humans and pigs), H9N2, H7N2 , H7N3 and H10N7. [00105] The Influenza virus B genus has one species, influenza B virus. Influenza B almost exclusively infects humans and is less common than influenza A. The only other animal known to be susceptible to influenza B infection is the seal. This type of influenza mutates at a rate 2-3 times slower than type A and consequently is less genetically diverse, with only one influenza B serotype. As a result of this lack of antigenic diversity, a degree of immunity to influenza B is usually acquired at an early age. However, influenza B mutates enough that lasting immunity is not possible. This reduced rate of antigenic change, combined with its limited host range (inhibiting cross species antigenic shift), ensures that pandemics of influenza B do not occur.

The Influenza virus C genus has one species, influenza C virus, which infects humans and pigs and can cause severe illness and local epidemics. However, influenza C is less common than the other types and usually seems to cause mild disease in children. [00107] Influenza A, B and C viruses are very similar in structure. The virus particle is 80-120 nanometers in diameter and usually roughly spherical, although filamentous forms can occur. Unusually for a virus, its genome is not a single piece of nucleic acid; instead, it contains seven or eight pieces of segmented negative-sense RNA. The Influenza A genome encodes 11 proteins: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), Ml, M2, NSl, NS2(NEP), PA, PBl, PB1-F2 and PB2.

HA and NA are large glycoproteins on the outside of the viral particles. HA is a lectin that mediates binding of the virus to target cells and entry of the viral genome into the target cell, while NA is involved in the release of progeny virus from infected cells, by cleaving sugars that bind the mature viral particles. Thus, these proteins have been targets for antiviral drugs. Furthermore, they are antigens to which antibodies can be raised. Influenza A viruses are classified into subtypes based on antibody responses to HA and NA, forming the basis of the H and N distinctions (vide supra) in, for example, H5N1. [00109] Influenza produces direct costs due to lost productivity and associated medical treatment, as well as indirect costs of preventative measures. In the United States, influenza is responsible for a total cost of over $10 billion per year, while it has been estimated that a future pandemic could cause hundreds of billions of dollars in direct and indirect costs. Preventative costs are also high. Governments worldwide have spent billions of U.S. dollars preparing and planning for a potential H5N1 avian influenza pandemic, with costs associated with purchasing drugs and vaccines as well as developing disaster drills and strategies for improved border controls.

Current treatment options for influenza include vaccination, and chemotherapy or chemoprophylaxis with anti-viral medications. Vaccination against influenza with an influenza vaccine is often recommended for high-risk groups, such as children and the elderly, or in people that have asthma, diabetes, or heart disease. However, it is possible to get vaccinated and still get influenza. The vaccine is reformulated each season for a few specific influenza strains but cannot possibly include all the strains actively infecting people in the world for that season. It takes about six months for the manufacturers to formulate and produce the millions of doses required to deal with the seasonal epidemics; occasionally, a new or overlooked strain becomes prominent during that time and infects people although they have been vaccinated (as by the H3N2 Fujian flu in the 2003-2004 influenza season). It is also possible to get infected just before vaccination and get sick with the very strain that the vaccine is supposed to prevent, as the vaccine takes about two weeks to become effective. [00111] Further, the effectiveness of these influenza vaccines is variable. Due to the high mutation rate of the virus, a particular influenza vaccine usually confers protection for no more than a few years. A vaccine formulated for one year may be ineffective in the following year, since the influenza virus changes rapidly over time, and different strains become dominant.

Also, because of the absence of RNA proofreading enzymes, the RNA- dependent RNA polymerase of influenza vRNA makes a single nucleotide insertion error roughly every 10 thousand nucleotides, which is the approximate length of the influenza vRNA. Hence, nearly every newly-manufactured influenza virus is a mutant — antigenic drift. The separation of the genome into eight separate segments of vRNA allows mixing or reassortment of vRNAs if more than one viral line has infected a single cell. The resulting rapid change in viral genetics produces antigenic shifts and allows the virus to infect new host species and quickly overcome protective immunity.

Antiviral drugs can also be used to treat influenza, with neuraminidase inhibitors being particularly effective, but viruses can develop resistance to the standard antiviral drugs.

PAPER

http://pubs.acs.org/doi/full/10.1021/acs.oprd.6b00063

Development of a Scalable Synthesis of an Azaindolyl-Pyrimidine Inhibitor of Influenza Virus Replication

Jianglin Liang^*, John E. Cochran, Warren A. Dorsch, Ioana Davies, and Michael P. Clark

Vertex Pharmaceuticals Incorporated, 50 Northern Avenue, Boston, Massachusetts 02210, United States

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.6b00063

Publication Date (Web): April 08, 2016

A scalable, asymmetric route for the synthesis of the influenza virus replication inhibitor 2 is presented. The key steps include an enzymatic desymmetrization of cis-1,3-cyclohexanediester in 99% yield and 96% ee, S_NAr displacement of a methanesulfinylpyrimidine, and a Curtius rearrangement to form a morpholinyl urea. This high-yielding route allowed us to rapidly synthesize hundreds of grams of 2 in 99% purity to support in vivo studies.

About Influenza

Often called “the flu,” seasonal influenza is caused by influenza viruses, which infect the respiratory tract.¹ The flu can result in seasonal epidemics² and can produce severe disease and high mortality in certain populations, such as the elderly.³ Each year, on average 5 to 20 percent of the U.S. population gets the flu⁴ resulting in more than 200,000 flu-related hospitalizations and 36,000 deaths.⁵The overall national economic burden of influenza-attributable illness for adults is $83.3 billion.⁵ Direct medical costs for influenza in adults totaled $8.7 billion including $4.5 billion for adult hospitalizations resulting from influenza-attributable illness.⁵ The treatment of the flu consists of antiviral medications that have been shown in clinical studies to shorten the disease and reduce the severity of symptoms if taken within two days of infection.⁶ There is a significant need for new medicines targeting flu that provide a wider treatment window, greater efficacy and faster onset of action.

About Vertex

Vertex is a global biotechnology company that aims to discover, develop and commercialize innovative medicines so people with serious diseases can lead better lives. In addition to our clinical development programs focused on cystic fibrosis, Vertex has more than a dozen ongoing research programs aimed at other serious and life-threatening diseases.

Founded in 1989 in Cambridge, Mass., Vertex today has research and development sites and commercial offices in the United States, Europe, Canada and Australia. For four years in a row, Science magazine has named Vertex one of its Top Employers in the life sciences. For additional information and the latest updates from the company, please visit www.vrtx.com.

Vertex’s press releases are available at www.vrtx.com.

SYNTHESIS COMING

WO-2010148197

http://www.google.co.in/patents/WO2010148197A1?cl=en

General Scheme 44 SIMILAR TO A POINT BUT NOT SAME

(a) Pd(PPh₃)₄ sodium carbonate, DME/water, reflux (b) meta-chloroperbenzoic acid, dichloromethane, rt. (c) 20a, tetrahydrofuran, 5O°C (d) trifluoroacetic acid, dichloromethane, rt.

SIMILAR NOT SAME

(e) morpholιne-4-carbonyl chloride, dimethylformamide, rt (f) sodium methoxide, methanol, rt.

Formation of 5-fluoro-3-[5-fluoro-4-(methylthio)pyrimidin-2-yl]-1-tosyl-lΗ- pyrrolo[2,3-b]pyridine (44b)

2-Chloro-5-fluoro-4-methylsulfanyl-pyrimidine (34.1 g, 191.0 mmol) , 5-fluoro-1-(p- tolylsulfonyl)-3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)pyrrolo[2,3-b]pyridine, 44a, (53.0 g, 127.3 mmol) and Na₂Cθ3 (40.5 g, 381.9 mmol) were dissolved in a mixture of DME (795 mL) and water (159 mL). The mixture was purged with nitrogen for 20 minutes and treated with Pd(PPh₃ )₄ (7.4 g, 6.6 mmol). After purging with nitrogen for another 20 minutes, the reaction was heated to reflux overnight, cooled to room temperature and diluted with water (60OmL). The resulting suspension was stirred at room temperature for 30 minutes and the precipitate was then collected by filtration, washed with water and acetonitrile and dried at 50 °C to afford 48.2 g of 5-fluoro-3-[5-fluoro-4-(methylthio)pyrimidin-2-yl]-1-tosyl-1H- pyrrolo[2,3-b]pyridine as a white solid.

¹H NMR (300 MHz, OMSO-d6) δ 8.70 – 8.58 (m, 2H), 8.54 – 8.41 (m, 2H), 8.09 (d, J = 8.4 Hz, 2H), 7.45 (d, J= 8.2 Hz, 2H), 2.76 (s, 3H), 2.36 (s, 3H).

Formation of 5-fluoro-3-[5-fluoro-4-(methylsulfinyl)pyrimidin-2-yl]-1- tosyl-1H-pyrrolo[2,3-b]pyridine (44c)

5-fluoro-3 – [5 -fluoro-4-(methylthio)pyrimidin-2-yl] – 1 -tosyl- 1 H-pyrrolo [2,3 – b]pyridine, 44b, (48.2 g, 111.5 mmol) was dissolved in dichloromethane (2.3 L) and treated portionwise with m-CPBA (27.5 g, 122.6 mmol) while keeping the temperature below 20 °C. After addition was complete, the reaction was stirred at room temperature for 2 hours, then treated with another portion of m-CPBA (1.9 g) and stirred for another hour. The reaction mixture was washed with 12% aqueuous K₂CO3 (2 x 1.0 L) and the organic layer was dried on Na₂SO₄ and concentrated in vacuo to provide 50 g of 5-fluoro-3-[5-fluoro-4- (methylsulfinyl)pyrimidin-2-yl]-1-tosyl-1H-pyrrolo[2,3-b]pyridine as a yellow solid.

¹H NMR (300 MHz, DMSO-rf<5) δ 9.11 (d, J= 1.5 Hz, 1H), 8.69 (s, 1H), 8.65 (dd, J = 9.0, 2.9 Hz, 1H), 8.52 (dd, J= 2.8, 1.2 Hz, 1H), 8.11 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.3 Hz, 2H), 3.05 (s, 3H), 2.36 (s, 3H).

[001057] Formation of tert-butyl N-[(IR, 3S)-3-[[5-fluoro-2-[5-fluoro-1-(p- tolylsulfonyl)pyrrolo [2,3-b] pyridin-3-yl]pyrimidin-4-yl] amino] cyclohexyl] carbamate (44d)

5-fluoro-3-(5-fluoro-4-methylsulfinyl-pyrimidin-2-yl)-1-(p-tolylsulfonyl)pyrrolo[2,3- b]pyridine, 44c, (5.9 g, 10.5 mmol) and tert-butyl N-[(IR, 35^*)-3-aminocyclohexyl]carbamate (3 g, 12.60 mmol) were dissolved in THF (100 mL). The reaction mixture was heated to 50 °C for 6 hours, then cooled to room temperature. C₆ lite was added and the solvent was removed under reduced pressure. The C₆ lite-supported residue was purified by silica gel chromatography (20-80% EtOAc/hexanes gradient to provide 3.7 g of tert-butyl N-[(IR, 3S)- 3-[[5-fluoro-2-[5-fluoro-1-(p-tolylsulfonyl)pyrrolo[2,3-b]pyridin-3-yl]pyrimidin-4- yl]amino]cyclohexyl]carbamate.

¹H NMR (300 MHz, CDCl₃) δ 8.51 (s, 1H), 8.46 – 8.41 (m, 1H), 8.29 (d, J = 1.6 Hz, 1H), 8.11 (s, 1H), 8.08 (s, 1H), 8.06 (d, J= 3.2 Hz, 1H), 7.27 (d, J= 8.4 Hz, 2H), 4.91 (d, J = 8.0 Hz, 1H), 4.41 (s, 1H), 4.29 – 4.01 (m, 1H), 3.64 (s, 1H), 2.47 (d, J= 11.5 Hz, 1H), 2.36 (s, 3H), 2.24 (d, J = 13.1 Hz, 1H), 2.08 (d, J= 10.9 Hz, 1H), 1.91 (d, J= 13.8 Hz, 1H), 1.43 (s, 9H), 1.30 – 1.03 (m, 4H).

Formation of (IS, SΛHVHS-fluoro^-β-fluoro-1-Cp- tolylsulfonyl)pyrrolo[2,3-b]pyridin-3-yl]pyrimidin-4-yl]cyclohexane-1,3-diamine (44e) tert-Butyl N-[(IR, 3S>3-[[5-fluoro-2-[5-fluoro-1-(p-tolylsulfonyl)pyrrolo[2,3- b]pyridin-3-yl]pyrimidin-4-yl]amino]cyclohexyl]carbamate, 44d, (3.7 g, 6.2 mmol) was dissolved in dichloromethane (105 mL) and treated with trifluoroacetic acid (31 mL). After 5 minutes, the volatiles were evaporated under reduced pressure, and the resulting residue was treated with IN NaOH (75 mL). The resulting precipitate was collected by filtration, washed with water (3 x 30 mL) and vacuum dried to provide 2.7 g of (IS, 3R)-Nl -[5-fluoro-2-[5- fluoro-1-(p-tolylsulfonyl)pyrrolo[2,3-b]pyridin-3-yl]pyrimidin-4-yl]cyclohexane-l,3-diamine as a white solid.

¹H NMR (300 MHz, MeOD) d 8.56 (dd, J = 8.0, 3.9 Hz, 2H), 8.35 – 8.26 (m, 1H), 8.12 (dd, J= 10.3, 6.1 Hz, 3H), 7.43 (d, J= 8.4 Hz, 2H), 4.36 – 4.21 (m, 1H), 3.28 – 3.13 (m, 1H), 2.48 (d, J= 12.3 Hz, 1H), 2.46 (s, 3H), 2.25 – 1.97 (m, J= 17.3, 10.6, 4.1 Hz, 4H), 1.76 – 1.28 (m, 3H).

Formation of N-[(IR, 3S>3-[[5-fluoro-2-[5-fluoro-1-(p- tolylsulfonyl)pyrrolo[2,3-b]pyridin-3-yl]pyrimidin-4-yl]amino]cyclohexyl] morpholine- 4-carboxamide (44f)

(15, 3R)-M-[5-fluoro-2-[5-fluoro-1-(p-tolylsulfonyl)pyrrolo[2,3-b]pyridin-3- yl]pyrimidin-4-yl]cyclohexane- 1,3 -diamine, 44e, (2.3 g, 4.6 mmol) was dissolved in DMF (5OmL) and treated with morpholine-4-carbonyl chloride (2.1 g, 13.8 mmol) and DIPEA (4.2 g, 5.6 mL, 32.3 mmol). After one hour, the resulting solution was diluted with water (400 mL) and stirred for an additional two hours. The resulting precipitate was collected by filtration, washed with water (3 x 50 mL) and dried to provide the crude product. This material was purified by flash chromatography on a 4Og column using EtOAc/DCM 20- 100%, to provide 2.0 g of N-[(1R, 35)-3-[[5-fluoro-2-[5-fluoro-1-(p- tolylsulfonyl)pyrrolo[2,3-b]pyridin-3-yl]pyrimidin-4-yl]amino]cyclohexyl]morpholine-4- carboxamide as a white solid.

¹H NMR (300 MHz, DMSO-Λ5) δ 8.53 – 8.43 (m, J = 11.9, 2.7 Hz, 3H), 8.22 (d, J = 3.9 Hz, 1H), 8.07 (d, J= 8.4 Hz, 2H), 7.44 (d, J= 8.3 Hz, 2H), 6.32 (d, J= 7.5 Hz, 1H), 4.05 (s, J= 19.4 Hz, 1H), 3.62 (s, 1H), 3.58 – 3.45 (m, 4H), 3.27 – 3.18 (m, 4H), 2.36 (s, 3H), 2.12 (d, J= 11.7 Hz, 1H), 1.99 (d, J= 9.5 Hz, 1H), 1.83 (d, J= 10.3 Hz, 2H), 1.53 – 1.11 (m, J = 32.3, 22.8, 10.9 Hz, 4H).

ormation of N-[(IR, 3S>3-[[5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3- b]pyridin-3-yl)pyrimidin-4-yl] amino] cyclohexyl]morpholine-4-carboxamide (706)

N- [( IR, 35)-3 – [ [5 -fluoro-2- [5 -fluoro- 1 -(p-tolylsulfonyl)pyrrolo [2,3 -b]pyridin-3 – yl]pyrimidin-4-yl]amino]cyclohexyl]morpholine-4-carboxamide, 44f, (2.0 g, 3.2 mmol) was suspended in methanol (50 mL) and treated with 25% sodium methoxide in methanol (19.9 mL, 92.3 mmol) . After stirring for 1 hour, the solvent was evaporated under reduced pressure, and the residue was partitioned between water (100 mL) and ethyl acetate (100 mL). The organic layer was collected, dried on Νa₂SO₄ and concentrated to provide the crude product as a yellow solid. This material was purified by silica gel chromatography on a 4Og column, using DCM/MeOH 1-6%. The purified fractions were treated with 2N HCl in ether and concentrated to provide 1.5 g of N-[(1R, 35)-3-[[5-fluoro-2-(5-fluoro-1H- pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl]amino]cyclohexyl]-morpholine-4-carboxamide as a white solid.

HCI D DCM

44e

Formation of (IS, S^-M-^-fluoro-S-CS-fluoro-1H-pyrrolo^S-^pyridin- 3-yl)phenyl)cyclohexane-1,3-diamine (44e)

To a solution of tert-butyl (IR, 35)-3-(2-fluoro-5-(5-fluoro-1-tosyl-lH-pyrrolo-[2,3- &]pyridin-3-yl)phenylamino)cyclohexylcarbamate, 44d, (0.65 g, 1.09 mmol) in methylene chloride (22 mL) was added hydrogen chloride (2.71 mL of 4M solution in 1,4-dioxane, 10.86 mmol). The reaction was heated to 50 °C and stirred for 6 hours. The mixture was cooled to room temperature and concentrated in vacuo, producing a yellow solid. The crude residue was purified via silica gel chromatography (25-50% Ethyl Acetate/hexanes gradient). Desired fractions were combined and concentrated in vacuo to produce 350 mg of 44e as a yellow powder.

General Scheme 67 SIMILAR TO A POINT BUT NOT SAME

(a) Pd/C (wet, Degussa), hydrogen, EtOH (b) 2,4-dichloro-5-fluoropyrimidine, ¹Pr₂NEt, THF, reflux (c) LiOH, THF/water, 5O°C

SIMILAR BUT NOT SAME

(d) DPPA, Et₃N, THF, 85 °C (e) 5-fluoro-3-(4,4,5,5-tetramethyl-1,3 ,2-dioxaborolan-2-yl)-1- tosyl-l//-pyrrolo[2,3-i]pyridine, XPhos, Pd₂(dba)₃, K₃PO₄, 2-methylTHF, water, 125 °C (f)

Formation (IR, 35)-ethyl 3-aminocyclohexanecarboxylate (67b)

To a solution of (IR, 35)-ethyl 3-(benzyloxycarbonylamino)cyclohexane-carboxylate, 18b, (14.0 g, 45.9 mmol) in ethanol (3 mL) was added Pd/C (wet, Degussa (2.4 g, 2.3 mmol). The mixture was evacuated and then stirred under atmosphere of nitrogen at room temperature overnight. The reaction mixture was filtered through a pad of celite and the resulting filtrate concentrated in vacuo to provide an oil that was used without further purification.

Formation (IR, SS^-ethyl 3-(2-chloro-5-fluoropyrimidin-4-ylamino)cyclohexane- carboxylate (67c)

To a solution of (IR, 3«S)-ethyl S-aminocyclohexanecarboxylate, 67b, (5.1 g, 24.1 mmol) and 2,4-dichloro-5,-fluoropyrimidine (6.0 g, 36.0 mmol) in THF (60 mL) was added diisopropylethylamine (9.6 mL, 55.4 mmol). The mixture was heated to reflux overnight. The reaction was cooled to room temperature and concentrated in vacuo. The residue was diluted with water and extracted twice with ethyl acetate. The combined organic phases were dried (MgSO₄), filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (0-40% EtOAc/hexanes gradient) to provide 6.7 g of (IR, 35^*)-ethyl 3-(2- chloro-5-fluoropyrimidin-4-ylamino)cyclohexane-carboxylate as a white solid: LCMS RT = 3.1 (M+H) 302.2.

Formation (IR, 35)-3-(2-chloro-5-fluoropyrimidin-4-ylamino)cyclohexanecarboxylic acid (67d)

To a solution of (IR, 35^*)-ethyl 3-(2-chloro-5-fluoropyrimidin-4- ylamino)cyclohexane-carboxylate, 67c, (20.0 g, 66.3 mmol) in THF (150 mL) was added added a solution of LiOH hydrate (8.3 g, 198.8 mmol) in 100ml water. The reaction mixture was stirred at 50 °C overnight, To the reaction mixture was added HCl (16.6 mL of 12 M solution, 198.8 mmol) and EtOAc. The organic phase was washed with brine and dried over MgSO₄ and the solvent was removed under reduced pressure to afford 17.5 g of product that was used without further purification: ¹H NMR (300 MHz, CDC13) δ 7.91 (d, J = 2.7 Hz, 2H), 5.24 (d, J = 7.3 Hz, 2H), 4.19 – 4.03 (m, 3H), 3.84 – 3.68 (m, 3H), 2.59 (ddd, J= 11.5, 8.2, 3.6 Hz, 2H), 2.38 (d, J = 12.4 Hz, 2H), 2.08 (d, J = 9.6 Hz, 6H), 1.99 – 1.76 (m, 5H), 1.63 – 1.34 (m, 6H), 1.32 – 1.15 (m, 4H).

Formation N-((1R, 35)-3-(2-chloro-5-fluoropyrimidin-4-ylamino)cyclohexyl)- pyrrolidine-1-carboxamide (67e)

A solution of (IR, 35)-3-(2-chloro-5-fluoropyrimidin-4-ylamino)cyclohexane- carboxylic acid, 67d, (8.2 g, 30.0 mmol), (azido(phenoxy)phosphoryl)oxybenzene (9.7 mL, 45.0 mmol) and triethylamine (5.8 mL, 42.0 mmol) in THF (200 mL) was degassed under nitrogen for 15 minutes. The reaction mixture was heated at 85 °C for 30 minutes until LC/MS indicated complete consumption of carboxylic acid, 67d. To the reaction mixture was added pyrrolidine (7.5 mL, 90.0 mmol) and the reaction was heated at 85 °C for an additional 15 min. The mixture was diluted into brine and extracted with EtOAc. The organic phase was separated, dried over MgSO₄. The product was isolated (6.25 g) by filtration after partial removal of solvent in vacuo: ¹H NMR (300 MHz, CDC13) δ 7.87 (d, J = 2.8 Hz, 2H), 5.04 (d, J = 8.1 Hz, 2H), 4.09 (ddd, J = 26.9, 13.4, 5.6 Hz, 4H), 3.91 – 3.71 (m, 2H), 3.32 (t, J= 6.5 Hz, 7H), 2.45 (d, J= 11.5 Hz, 2H), 2.08 (dd, J= 22.1, 12.0 Hz, 4H), 1.96- 1.82 (m, 9H), 1.54 (dd, J= 18.6, 8.5 Hz, 2H), 1.22 – 1.01 (m, 6H).

Formation N-((IR, 3S>3-(5-fluoro-2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridm-3- yl)pyrimidin-4-ylamino)cyclohexyl)pyrrolidine-1-carboxamide (67f)

A solution of N-((1R, 3«S)-3-(2-chloro-5-fluoropyrimidin-4-ylamino)cyclohexyl)- pyrrolidine-1-carboxamide, 67e, (6.8 g, 20.0 mmol), 5-fluoro-1-(p-tolylsulfonyl)-3-(4,4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)pyrrolo[2,3-b]pyridine, 44a, (12.5 g, 30.0 mmol) and K₃PO₄ (17.0 g, 80.0 mmol) in 2-methyl TΗF (180 mL) and water (20 mL) was degassed under nitrogen for 30 min. To the mixture was added dicyclohexyl-[2-(2,4,6- triisopropylphenyl)phenyl]phosphane (XPhos) (1.1 g, 2.4 mmol) and Pd₂(dba)₃ (0.5 g, 0.5 mmol). The reaction mixture was heated in a pressure bottle at 125 °C for 2.5 hr. The reaction mixture was filtered through celite, the solvent was removed under reduced pressure. The resulting residue was purified by silica gel chromatography (8%MeOΗ/CΗ₂Cl₂) to afford 11.5 g of the desired product: ¹H ΝMR (300 MHz, CDC13) δ 8.54 (s, 1H), 8.49 (dd, J= 9.0, 2.8 Hz, 1H), 8.32 (d, J= 2.1 Hz, 1H), 8.13 (d, J= 8.3 Hz, 2H), 8.07 (d, J= 3.2 Hz, 1H), 7.30 (d, J = 8.5 Hz, 2H), 4.98 (d, J = 6.3 Hz, 1H), 4.37 – 4.16 (m, 1H), 4.08 (d, J = 7.3 Hz, 1H), 3.99 – 3.80 (m, 1H), 3.33 (t, J= 6.5 Hz, 4H), 2.52 (d, J= 11.6 Hz, 1H), 2.39 (s, 3H), 2.29 (d, J= 11.3 Hz, 1H), 2.12 (d, J= 11.1 Hz, 1H), 1.99 – 1.81 (m, 5H), 1.70 – 1.55 (m, 1H), 1.22 – 1.08 (m, 2H).

Formation N-((IR, 3S>3-(5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)- pyrimidin-4-ylamino)cyclohexyl)pyrrolidine-1-carboxamide (895)

A solution of N-((1R, 35)-3-(5-fluoro-2-(5-fluoro-1-tosyl-lH-pyrrolo[2,3-b]pyridin-3- yl)pyrimidin-4-ylamino)cyclohexyl)pyrrolidine-1-carboxamide, 67f, (11.5 g, 19.3 mmol) in TΗF (150 mL) was added sodium methoxide (4.173 g, 19.31 mmol). After stirring the reaction mixture for 2 minutes, the mixture was poured into an aqueous saturated solution of NaHCO₃. The organic phase was washed with brine, dried over MgSO₄ and the solvent was removed under reduced pressure. The resulting residue was purified by silica gel chromatography (10%MeOH/CH₂Cl₂) to afford 6.5g of the desired product. The product was converted to an HCl salt by dissolving in MeOH (100 mL) and adding 2.4 mL of 12M HCl solution at room temperature. The solution was stirred at for lhour and the HCl salt precipitated out and filtered to provide 7.05g of the HCl salt: ¹H NMR (300 MHz, DMSO) δ 9.36 (s, 2H), 9.05 (d, J= 3.0 Hz, 2H), 8.49 (d, J= 5.6 Hz, 2H), 8.41 (dd, J= 2.6, 1.4 Hz, 2H), 8.31 (d, J= 9.5 Hz, 2H), 5.92 (s, 3H), 4.24 (s, 3H), 3.64 (s, 2H), 3.18 (t, J= 6.6 Hz, 7H), 2.07 (dt, J = 22.7, 11.5 Hz, 4H), 1.87 (t, J = 12.6 Hz, 4H), 1.77 (dd, J = 8.0, 5.3 Hz, 7H), 1.65 – 1.13 (m, 8H).

PATENT

US-20120171245-A1 / 2012-07-05