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

Optimization, Production, and Characterization of a CpG-Oligonucleotide-Ficoll Conjugate Nanoparticle Adjuvant for Enhanced Immunogenicity of Anthrax Protective Antigen

Uncategorized Comments Off

May 132016

We have synthesized and characterized a novel phosphorothioate CpG oligodeoxynucleotide (CpG ODN)-Ficoll conjugated nanoparticulate adjuvant, termed DV230-Ficoll. This adjuvant was constructed from an amine-functionalized-Ficoll, a heterobifunctional linker (succinimidyl-[(N-maleimidopropionamido)-hexaethylene glycol] ester) and the CpG-ODN DV230. Herein, we describe the evaluation of the purity and reactivity of linkers of different lengths for CpG-ODN-Ficoll conjugation, optimization of linker coupling, and conjugation of thiol-functionalized CpG to maleimide-functionalized Ficoll and process scale-up. Physicochemical characterization of independently produced lots of DV230-Ficoll reveal a bioconjugate with a particle size of approximately 50 nm and covalent attachment of more than 100 molecules of CpG per Ficoll. Solutions of purified DV230-Ficoll were stable for at least 12 months at frozen and refrigerated temperatures and stability was further enhanced in lyophilized form. Compared to nonconjugated monomeric DV230, the DV230-Ficoll conjugate demonstrated improved in vitro potency for induction of IFN-α from human peripheral blood mononuclear cells and induced higher titer neutralizing antibody responses against coadministered anthrax recombinant protective antigen in mice. The processes described here establish a reproducible and robust process for the synthesis of a novel, size-controlled, and stable CpG-ODN nanoparticle adjuvant suitable for manufacture and use in vaccines.

READ……http://pubs.acs.org/doi/full/10.1021/acs.bioconjchem.6b00107

Optimization, Production, and Characterization of a CpG-Oligonucleotide-Ficoll Conjugate Nanoparticle Adjuvant for Enhanced Immunogenicity of Anthrax Protective Antigen

Bob Milley^*^†, Radwan Kiwan^†, Gary S. Ott^†, Carlo Calacsan^†, Melissa Kachura^†, John D. Campbell^†, Holger Kanzler^‡, and Robert L. Coffman^†

^† Dynavax Technologies Corporation, 2929 Seventh Street, Suite 100, Berkeley, California 94710, United States

^‡ MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland 20878, United States

Bioconjugate Chem., Article ASAP

DOI: 10.1021/acs.bioconjchem.6b00107

Publication Date (Web): April 13, 2016

*E-mail: bmilley@dynavax.com. Phone: (510) 665-7227. Fax: (510) 848-1327.

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.

//////Optimization, Production, Characterization, CpG-Oligonucleotide-Ficoll Conjugate Nanoparticle Adjuvant, Enhanced Immunogenicity, Anthrax Protective Antigen

Solvates, Salts, and Cocrystals: A Proposal for a Feasible Classification System

Uncategorized Comments Off

May 132016

The design of pharmaceutical cocrystals has initiated widespread debate on the classification of cocrystals. Current attempts to classify multicomponent crystals suffer from ambiguity, which has led to inconsistent definitions for cocrystals and for multicomponent crystals in general. Inspired by the work of Aitipamula et al. (Cryst. Growth Des. 2012, 12, 2147–2152), we present a feasible classification system for all multicomponent crystals. The present classification enables us to analyze and classify multicomponent crystal structures present in the Cambridge Structural Database (CSD). This reveals that all seven classes proposed are relevant in terms of frequency of occurrence. Lists of CSD refcodes for all classes are provided. We identified over 5000 cocrystals in the CSD, as well as over 12 000 crystals with more than two components. This illustrates that the possibilities for alternative drug formulations can be increased significantly by considering more than two components in drug design.

READ ………….http://pubs.acs.org/doi/full/10.1021/acs.cgd.6b00200

Solvates, Salts, and Cocrystals: A Proposal for a Feasible Classification System

E. Grothe^†, H. Meekes^†, E. Vlieg^†, J. H. ter Horst^‡, and R. de Gelder^*^†

^† Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands

^‡ EPSRC Centre for Innovative Manufacturing in Continuous Manufacturing and Crystallisation, Strathclyde Institute of Pharmacy and Biomedical Sciences, Technology and Innovation Centre, University of Strathclyde, 99 George Street, Glasgow G1 1RD, United Kingdom

Cryst. Growth Des., Article ASAP

DOI: 10.1021/acs.cgd.6b00200

Publication Date (Web): April 21, 2016

*E-mail: r.degelder@science.ru.nl.

Synopsis

A classification system for multicomponent crystals is applied to the organic crystals in the Cambridge Structural Database (CSD) in an attempt to reveal the population of structures within each subclass. Seven subclasses of multicomponent crystals are presented, each illustrated by an example of an isonicotinamide crystal structure that can be found in the CSD.

//////////Solvates, Salts, Cocrystals, Proposal, Feasible Classification System

Teslaphoresis of Carbon Nanotubes

Uncategorized Comments Off

May 132016

This paper introduces Teslaphoresis, the directed motion and self-assembly of matter by a Tesla coil, and studies this electrokinetic phenomenon using single-walled carbon nanotubes (CNTs). Conventional directed self-assembly of matter using electric fields has been restricted to small scale structures, but with Teslaphoresis, we exceed this limitation by using the Tesla coil’s antenna to create a gradient high-voltage force field that projects into free space. CNTs placed within the Teslaphoretic (TEP) field polarize and self-assemble into wires that span from the nanoscale to the macroscale, the longest thus far being 15 cm. We show that the TEP field not only directs the self-assembly of long nanotube wires at remote distances (>30 cm) but can also wirelessly power nanotube-based LED circuits. Furthermore, individualized CNTs self-organize to form long parallel arrays with high fidelity alignment to the TEP field. Thus, Teslaphoresis is effective for directed self-assembly from the bottom-up to the macroscale.

SEE………..http://pubs.acs.org/doi/full/10.1021/acsnano.6b02313

Teslaphoresis of Carbon Nanotubes

^†Department of Chemistry, ^‡Department of Materials Science and NanoEngineering, ^§Smalley-Curl Institute, Rice University, 6100 Main Street, Houston, Texas 77005, United States

^∥ Department of Chemistry and Physics, University of Tennessee—Chattanooga, 615 McCallie Avenue, Chattanooga, Tennessee 37403, United States

^⊥ Department of Biomedical Engineering, Texas A&M University, 101 Bizzell Street, College Station, Texas 77843,United States

^# Second Baptist School, 6410 Woodway Drive, Houston, Texas 77057, United States

ACS Nano, 2016, 10 (4), pp 4873–4881

DOI: 10.1021/acsnano.6b02313

Publication Date (Web): April 13, 2016

*E-mail: paul.cherukuri@rice.edu.

Keywords:

dielectrophoresis, directed self-assembly, carbon nanotubes, Tesla, wireless energy

/////////

Palladium- and Nickel-Catalyzed Amination of Aryl Fluorosulfonates

spectroscopy, SYNTHESIS Comments Off

May 132016

Examples of the palladium- and nickel-catalyzed amination of aryl fluorosulfonates with aromatic and alkyl amines are described. Aniline is coupled to a diverse series of aryl fluorosulfonates catalyzed by the combination of CpPd(cinammyl) and Xantphos, and the relative reactivity of aryl fluorosulfonates to undergo Pd-catalyzed amination was compared with other common aryl electrophiles. In addition, we report the direct amination of a phenol by in situ formation of an aryl fluorosulfonate by reaction with sulfuryl fluoride and base followed by subsequent amination to form a new C–N bond. Finally, we report examples of the nickel-catalyzed amination of aryl fluorosulfonates catalyzed by the combination of Ni(COD)₂ and DPPF in the presence of MeCN. The high reactivity of the aryl fluorosulfonate electrophile with generic palladium and nickel catalyst systems, combined with its simple preparation from sulfuryl fluoride will enable commercial amination reactions of abundant phenolic raw materials.

SEE…….http://pubs.acs.org/doi/full/10.1021/acscatal.6b00865

Palladium- and Nickel-Catalyzed Amination of Aryl Fluorosulfonates

Patrick S. Hanley^*, Thomas P. Clark, Arkady L. Krasovskiy, Matthias S. Ober, John P. O’Brien, and Tina S. Staton

The Dow Chemical Company, Core R&D, Midland, Michigan 48674, United States

ACS Catal., 2016, 6, pp 3515–3519

DOI: 10.1021/acscatal.6b00865

Publication Date (Web): April 27, 2016

*E-mail: pshanley@dow.com.

////Palladium, Nickel-Catalyzed , Amination , Aryl Fluorosulfonates

Dihydrobenzofuran Neolignans

Uncategorized Comments Off

May 132016

Figure 1 Structures of dihydrobenzofuran neolignans 2a and 2b.

Scheme 1 (i) Ag₂O, (CH₃)₂CO:C₆H₆ 3:5, r.t., 20 h (2a: 36% yield; 2b: 43% yield).

Figure 3 Main nuclear Overhauser effect (NOE) correlations observed in the nuclear Overhauser effect spectroscopy (NOESY) spectra of compounds 2a and 2b.

Table 1 ¹H and ¹³C NMR data assignments for compound 2a (400 MHz, CDCl₃)

	δ_Ca	δ_H (integral, multiplicityb), J/ Hz
1	132.0 (C)	−
2=6	127.5 (CH)	7.27 (2H, ddd, J_2,5 = J_6,3 0.3, J_2,7 = J_6,7 0.6, J_2,3 = J_6,5 8.3)
3=5	115.7 (CH)	6.84 (2H, dd, J_3,6 = J_5,2 0.3, J_3,2 = J_5,6 8.3)
4	156.1 (C)	−
7	87.7 (CH)	6.09 (1H, dt, J_7,2 = J_7,6 0.6, J_7,8 7.2)
8	55.1 (CH)	4.27 (1H, dd, J_8,6′ 1.4, J_8,7 7.2)
9	170.9 (C)	−
10	52.9 (CH₃)	3.83 (3H, s)
1′	127.8 (C)	−
2′	130.8 (CH)	7.43 (1H, ddd, J_2′,7‘ 1.1, J_2′,6′ 2.0, J_2′,3′ 8.3)
3′	110.3 (CH)	6.89 (1H, dd, J_3′,6′ 0.4, J_3′,2′8.3)
4′	161.2 (C)	−
5′	125.1 (C)	−
6′	124.9 (CH)	7.55 (1H, dddd, J_6′,3′ 0.4, J_6′,7′ 0.7, J_6′,8 1.4, J_6′,2′2.0)
7′	144.7 (CH)	7.66 (1H, ddd, J_7′,6′ 0.7, J_7′,2′ 1.1, J_7′,8′ 15.9)
8′	115.2 (CH)	6.32 (1H, d, J_8′,7′ 15.9)
9′	167.9 (C)	−
10′	51.7 (CH₃)	3.81 (3H, s)

^aMultiplicities assigned on the basis of distortionless enhancement by polarization transfer (DEPT) 135 experiments;

^bmultiplicities and coupling constant values measured within ¹H NMR and J-resolved spectra with the help from¹H-¹H correlation spectroscopy (COSY) results.

Table 2 2D NMR data for compound 2a (400 MHz, CDCl₃)

C	H	gCOSYa	gHMBCb	gHMQCc	NOESYd
1	–	–	H₃ =H₅, H₇, H₈	–	–
2=6	2=6	H₃, H₅, H₇	H₃ =H₅, H₇	H₂=H₆	H₇, H₈
3=5	3=5	H₂, H₆	H₅	H₃=H₅	–
4	–	–	H₃ =H₅, H₂ =H₆	–	–
7	7	H₂=H₆, H₈	H₆, H₈	H₇	H₂=H₆*
8	8	H₇, H_6′	H₇, H_6′	H₈	H₂=H₆, H_6′*
9	–	–	H₇, H₈, H₁₀	–	–
10	10	–	−	H₁₀	–
1′	–	–	H_3′, H_8′	–	–
2′	2′	H_3′, H_6′, H_7′	H_6′, H_7′	H_2′	H_7′, H_8′*
3′	3′	H_2′, H_6′	−	H_3′	–
4′	–	–	H_2′, H_3′, H_6′, H₇, H₈	–	–
5′	–	–	H₈, H_3′	–	–
6′	6′	H_2′, H_3′, H_7′, H₈	H_2′, H_7′, H₈	H_6′	H_8′, H_7′, H₈*
7′	7′	H_2′, H_8′, H_6′	H_2′, H_6′, H_8′	H_7′	H_6′*, H_2′
8′	8′	_H7′	H_7′	H_8′	H_6′*, H_2′
9′	–	–	H_7′, H_8′, H_10′	–	–
10′	10′	–	–	H_10′	–

^bgradient-selected heteronuclear multiple bond coherence;
^cgradient-selected heteronuclear multiple quantum coherence;
^dnuclear Overhauser effect spectroscopy.
^*mean weak correlation.

^aGradient-selected correlation spectroscopy;

Table 3 ¹H and ¹³C NMR data assignments for compound 2b (400 MHz, acetone-d₆)

	δ_Ca	δ_H (integral, multiplicityb); J/ Hz
1	132.5 (C)	–
2	111.2 (CH)	7.10 (1H, ddd, J_2,5 0.3, J_2,7 0.8, J_2,6 2.1)
3	149.1 (C)	–
4	148.5 (C)	–
5	116.3 (CH)	6.84 (1H, dd, J_5,2 0.3, J_5,6 8.3)
6	120.7 (CH)	6.92 (1H, ddd, J_6,7 0.6, J_6,2 2.1, J_6,5 8.3)
7	88.8 (CH)	6.04 (1H, ddd, J_7,6 0.6, J_7,2 0.8, J_7,8 7.3)
8	57.0 (CH)	4.47 (1H, dd, J_8,6′ 1.4, J_8,7 7.3)
9	172.1 (C=O)	–
10	53.5 (CH₃)	3.81 (3H, s)
11	56.4 (CH₃)	3.84 (3H, s)
1′	129.9 (C)	–
2′	113.9 (CH)	7.33 (1H, dd, J_2′,7′ 0.4, J_2’6′ 2.6)
3′	146.3 (C)	–
4′	151.5 (C)	–
5′	127.8 (C)	–
6′	119.5 (CH)	7.29 (1H, ddd, J_6′,7′ 0.8, J_6′,8 1.4, J_6′,2′ 2.6)
7′	145.9 (CH)	7.63 (1H, ddd, J_7′,2′ 0.4, J_7′,6′ 0.8, J_7′,8′ 15.8)
8′	116.8 (CH)	6.44 (1H, d, J_8′,7′ 15.8)
9′	168.2 (C)	–
10′	52.1 (CH₃)	3.73 (3H, s)
11′	56.8 (CH₃)	3.92 (3H, s)

^aMultiplicities assigned on the basis of distortionless enhancement by polarization transfer (DEPT) 135 experiments;
^bmultiplicities and coupling constant values measured within ¹H-NMR and J-resolved spectra with the help from¹H-¹H correlation spectroscopy (COSY) results.

Table 4 2D NMR data for compound 2b (400 MHz, acetone-d₆)

C	H	gCOSYa	gHMBCb	gHMQCc	NOESYd
1	–	–	H₂, H₆, H₇, H₈	–	–
2	2	H₅, H₆, H₇	H₅, H₆, H₇	H₂	H₇, H₈, H₁₁
3	–	–	H₂, H₅, H₁₁	H₃	–
4	–	–	H₂, H₅, H₆	–	–
5	5	H₂, H₆	H₆	H₅	–
6	6	H₂, H₅, H₇	H₂, H₅	H₆	H₇, H₈
7	7	H₂, H₆, H₈	H₂, H₆, H₈	H₇	H₆*, H₂
8	8	H_6′, H₇	H₂, H_6′	H₈	H_6′*, H₂, H₆
9	–	–	H₇, H₈, H₁₀	H₉	–
10	10	–	−	H₁₀	–
11	11	–	−	H₁₁	H₂
1′	–	–	H_7′, H_8′	H_1′	–
2′	2′	H_6′, H_7′	H_6′, H_7′	H_2′	H_7′, H_8′*, H_11′
3′	–	–	H_11′	–	–
4′	–	–	H_2′, H_6′, H₇, H₈	–	–
5′	–	–	H₇, H_8′	–	–
6′	6′	H_7′, H_2′, H₈	H_2′, H_7′, H₈	H_6′	H_8′, H_7′, H₈*
7′	7′	H_2′, H_6′, H_8′	H_2′, H_6′, H_8′	H_7′	H_6′, H_2′,
8′	8′	H_7′	H_7′	H_8′	H_6′, H_2′
9′	–	–	H_8′, H_10′	–	–
10′	10′	–	–	H_10′	–
11′	11′	–	–	H_11′	H_2′

^aGradient-selected correlation spectroscopy;
^bgradient-selected heteronuclear multiple bond coherence;
^cgradient-selected heteronuclear multiple quantum coherence;
^dnuclear Overhauser effect spectroscopy.
^*mean weak correlation.

¹H and ¹³C NMR data previously reported for compound2a and 2b were obtained in CDCl₃ or acetone-d₆. Most of the signals in the ¹H NMR spectrum were between δ_H 6.0 and δ_H 8.0, but the hydrogen signal multiplicities are ambiguous. In this work, we found that for compound2a in acetone-d₆, the signals at δ_H 7.6-7.7 are referred to four hydrogen atoms and their overlapping precluded their correct assignment (Figure 2). Therefore, CDCl₃ provided much clearer spectra for 2a, but not for 2b, due to the solvent influence on chemical shifts. For compound 2b, three hydrogen atoms resonate at δ_H 6.91 in the ¹H HMR spectrum in CDCl₃. On the other hand, the ¹H NMR signals of 2b were resolved by using acetone-d₆ as solvent, which allowed verification of the multiplicities, observation of the chemical shifts and measurement of the coupling constants.

Figure 2 Expansions of the ¹H NMR spectrum of compounds 2a and 2b obtained in CDCl₃ and acetone-d₆.

Journal of the Brazilian Chemical Society

On-line version ISSN 1678-4790

J. Braz. Chem. Soc. vol.27 no.1 São Paulo Jan. 2016

http://dx.doi.org/10.5935/0103-5053.20150262

ARTICLES

Detailed ¹H and ¹³C NMR Spectral Data Assignment for Two Dihydrobenzofuran Neolignans

Talita C. T. Medeiros^a^#, Herbert J. Dias^a^#, Eliane O. Silva^a, Murilo J. Fukui^b, Ana Carolina F. Soares^b, Tapas Kar^c, Vladimir C. G. Heleno^b, Paulo M. Donate^a, Renato L. T. Parreira^b, Antônio E. M. Crotti^a^*

^{^a}Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901 Ribeirão Preto-SP, Brazil

^{^b}Núcleo de Pesquisas em Ciências Exatas e Tecnológicas, Universidade de Franca, 14404-600 Franca-SP, Brazil

^{^c}Department of Chemistry and Biochemistry, Utah State University, 84322-0300 Logan-UT, United States

ABSTRACT

In this work we present a complete proton (1H) and carbon 13 (¹³C) nuclear magnetic resonance (NMR) spectral analysis of two synthetic dihydrofuran neolignans (±)-trans-dehydrodicoumarate dimethyl ester and (±)-trans-dehydrodiferulate dimethyl ester. Unequivocal assignments were achieved by ¹H NMR, proton decoupled ¹³C (¹³C{¹H}) NMR spectra, gradient-selected correlation spectroscopy (gCOSY), J-resolved, gradient-selected heteronuclear multiple quantum coherence (gHMQC), gradient-selected heteronuclear multiple bond coherence (gHMBC) and nuclear Overhauser effect spectroscopy (NOESY) experiments. All hydrogen coupling constants were measured, clarifying all the hydrogen signals multiplicities. Computational methods were also used to simulate the ¹H and ¹³C chemical shifts and showed good agreement with the transconfiguration of the substituents at C₇ and C₈.

http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-50532016000100136&lng=en&nrm=iso&tlng=en

Key words: neolignans, oxidative coupling, J-resolved, benzofurans

see……….0103-5053-jbchs-27-01-0136-suppl01.pdf

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Printing with Collagen

drugs Comments Off

May 132016

Printing with Collagen

Addition of collagen to hydrogels in 3D printing improves stem cell differentiation in osteogenesis

http://www.chemistryviews.org/details/news/9268521/Printing_with_Collagen.html

Bioprinting Organotypic Hydrogels with Improved Mesenchymal Stem Cell Remodeling and Mineralization Properties for Bone Tissue Engineering,
Daniela Filipa Duarte Campos, Andreas Blaeser, Kate Buellesbach, Kshama Shree Sen, Weiwei Xun, Walter Tillmann, Horst Fischer,
Adv. Healthcare Mater. 2016.
DOI: 10.1002/adhm.201501033

Collagen

Tropocollagen molecule: three left-handed procollagens (red, green, blue) join to form a right handed triple helical tropocollagen.

Collagen is the most common protein found in mammals.

Collagen /ˈkɒlədʒᵻn/ is the main structural protein in the extracellular space in the various connective tissues in animal bodies. As the main component of connective tissue, it is the most abundant protein in mammals,^[1] making up from 25% to 35% of the whole-body protein content. Depending upon the degree of mineralization, collagen tissues may be rigid (bone), compliant (tendon), or have a gradient from rigid to compliant (cartilage).^[2] Collagen, in the form of elongated fibrils, is mostly found in fibrous tissues such as tendons, ligaments and skin. It is also abundant incorneas, cartilage, bones, blood vessels, the gut, intervertebral discs and the dentin in teeth.^[3] In muscle tissue, it serves as a major component of the endomysium. Collagen constitutes one to two percent of muscle tissue, and accounts for 6% of the weight of strong, tendinous muscles.^[4] Thefibroblast is the most common cell that creates collagen.

Gelatin, which is used in food and industry, is collagen that has been irreversibly hydrolyzed.^[5] Collagen also has many medical uses in treating complications of the bones and skin.

The name collagen comes from the Greek κόλλα (kólla), meaning “glue“, and suffix -γέν, -gen, denoting “producing”.^[6]^[7] This refers to the compound’s early use in the process of boiling the skin and sinews of horses and other animals to obtain glue.

Collagen injections can be used in cosmetic procedures to improve the contours of aging skin.

Types of collagen

Collagen occurs in many places throughout the body. Over 90% of the collagen in the human body, however, is type I.^[8]

So far, 28 types of collagen have been identified and described. They can be divided into several groups according to the structure they form:^[2]

Fibrillar (Type I, II, III, V, XI)
Non-fibrillar
- FACIT (Fibril Associated Collagens with Interrupted Triple Helices) (Type IX, XII, XIV, XVI, XIX)
- Short chain (Type VIII, X)
- Basement membrane (Type IV)
- Multiplexin (Multiple Triple Helix domains with Interruptions) (Type XV, XVIII)
- MACIT (Membrane Associated Collagens with Interrupted Triple Helices) (Type XIII, XVII)
- Other (Type VI, VII)

The five most common types are:

Type I: skin, tendon, vascular ligature, organs, bone (main component of the organic part of bone)
Type II: cartilage (main collagenous component of cartilage)
Type III: reticulate (main component of reticular fibers), commonly found alongside type I.
Type IV: forms basal lamina, the epithelium-secreted layer of the basement membrane.
Type V: cell surfaces, hair and placenta

wrinkled mouth with cigarette
Tobacco contains chemicals that damage collagen

Medical uses

Cardiac applications

The collagenous cardiac skeleton which includes the four heart valve rings, is histologically and uniquely bound to cardiac muscle. The cardiac skeleton also includes the separating septa of the heart chambers – the interventricular septum and the atrioventricular septum. Collagen contribution to the measure of cardiac performance summarily represents a continuous torsional force opposed to the fluid mechanics of blood pressure emitted from the heart. The collagenous structure that divides the upper chambers of the heart from the lower chambers is an impermeable membrane that excludes both blood and electrical impulses through typical physiological means. With support from collagen, atrial fibrillation should never deteriorate to ventricular fibrillation. Collagen is layered in variable densities with cardiac muscle mass. The mass, distribution, age and density of collagen all contribute to the compliance required to move blood back and forth. Individual cardiac valvular leaflets are folded into shape by specialized collagen under variable pressure. Gradual calcium deposition within collagen occurs as a natural function of aging. Calcified points within collagen matrices show contrast in a moving display of blood and muscle, enabling methods of cardiac imaging technology to arrive at ratios essentially stating blood in (cardiac input) and blood out (cardiac output). Pathology of the collagen underpinning of the heart is understood within the category of connective tissue disease.

Hydrolyzed type II collagen and osteoarthritis

A published study^[9] reports that ingestion of a novel low molecular weight hydrolyzed chicken sternal cartilage extract, containing a matrix of hydrolyzed type II collagen,chondroitin sulfate, and hyaluronic acid, relieves joint discomfort associated with osteoarthritis. A randomized controlled trial (RCT) enrolling 80 subjects demonstrated that it was well tolerated with no serious adverse event and led to a significant improvement in joint mobility compared to the placebo group on days 35 (p = 0.007) and 70 (p < 0.001).

Fast facts on collagen

Here are some key points about collagen. More detail and supporting information is in the main article.^25-27

Protein makes up around 20% of the body’s mass, and collagen makes up around 30% of the protein in the human body.
There are at least 16 types of collagen, but 80-90% of the collagen in the body consists of types I, II, and III.
Type I collagen fibrils are stronger than steel (gram for gram).
Collagen is most commonly found within the body in the skin, bones and connective tissues.
The word “collagen” is derived from the Greek “kolla,” meaning glue.
Collagen gives the skin its strength and structure, and also plays a role in the replacement of dead skin cells.
Collagen production declines with age (as part of intrinsic aging), and is reduced by exposure to ultraviolet light and other environmental factors (extrinsic aging).
Collagen in medical products can be derived from human, bovine, porcine and ovine sources.
Collagen dressings attract new skin cells to wound sites.
Cosmetic products such as revitalizing lotions that claim to increase collagen levels are unlikely to do so, as collagen molecules are too large to be absorbed through the skin.
Collagen production can be stimulated through the use of laser therapy and the use of all-trans retinoic acid (a form ofvitamin A).
Controllable factors that damage the production of collagen include sunlight, smoking and high sugar consumption.

Cosmetic surgery

Collagen has been widely used in cosmetic surgery, as a healing aid for burn patients for reconstruction of bone and a wide variety of dental, orthopedic, and surgical purposes. Both human and bovine collagen is widely used as dermal fillers for treatment of wrinkles and skin aging.^[10] Some points of interest are:

When used cosmetically, there is a chance of allergic reactions causing prolonged redness; however, this can be virtually eliminated by simple and inconspicuous patch testing prior to cosmetic use.
Most medical collagen is derived from young beef cattle (bovine) from certified BSE-free animals. Most manufacturers use donor animals from either “closed herds”, or from countries which have never had a reported case of BSE such as Australia, Brazil, and New Zealand.

Bone grafts

As the skeleton forms the structure of the body, it is vital that it maintains its strength, even after breaks and injuries. Collagen is used in bone grafting as it has a triple helical structure, making it a very strong molecule. It is ideal for use in bones, as it does not compromise the structural integrity of the skeleton. The triple helical structure of collagen prevents it from being broken down by enzymes, it enables adhesiveness of cells and it is important for the proper assembly of the extracellular matrix.^[11]

Tissue regeneration

Collagen scaffolds are used in tissue regeneration, whether in sponges, thin sheets, or gels. Collagen has the correct properties for tissue regeneration such as pore structure, permeability, hydrophilicity and it is stable in vivo. Collagen scaffolds are also ideal for the deposition of cells, such as osteoblasts and fibroblasts and once inserted, growth is able to continue as normal in the tissue.^[12]

Reconstructive surgical uses

Collagens are widely employed in the construction of the artificial skin substitutes used in the management of severe burns. These collagens may be derived from bovine, equine, porcine, or even human sources; and are sometimes used in combination with silicones, glycosaminoglycans, fibroblasts, growth factors and other substances.

Collagen is also sold commercially in pill form as a supplement to aid joint mobility. However, because proteins are broken down into amino acids before absorption, there is no reason for orally ingested collagen to affect connective tissue in the body, except through the effect of individual amino acid supplementation.

Collagen is also frequently used in scientific research applications for cell culture, studying cell behavior and cellular interactions with the extracellular environment.^[13]

Wound care

Collagen is one of the body’s key natural resources and a component of skin tissue that can benefit all stages of the wound healing process.^[14] When collagen is made available to the wound bed, closure can occur. Wound deterioration, followed sometimes by procedures such as amputation, can thus be avoided.

Collagen is a natural product, therefore it is used as a natural wound dressing and has properties that artificial wound dressings do not have. It is resistant against bacteria, which is of vital importance in a wound dressing. It helps to keep the wound sterile, because of its natural ability to fight infection. When collagen is used as a burn dressing, healthygranulation tissue is able to form very quickly over the burn, helping it to heal rapidly.^[15]

Throughout the 4 phases of wound healing, collagen performs the following functions in wound healing:

Guiding function: Collagen fibers serve to guide fibroblasts. Fibroblasts migrate along a connective tissue matrix.
Chemotactic properties: The large surface area available on collagen fibers can attract fibrogenic cells which help in healing.
Nucleation: Collagen, in the presence of certain neutral salt molecules can act as a nucleating agent causing formation of fibrillar structures. A collagen wound dressing might serve as a guide for orienting new collagen deposition and capillary growth.
Hemostatic properties: Blood platelets interact with the collagen to make a hemostatic plug.

Chemistry

The collagen protein is composed of a triple helix, which generally consists of two identical chains (α1) and an additional chain that differs slightly in its chemical composition (α2).^[16] The amino acid composition of collagen is atypical for proteins, particularly with respect to its high hydroxyproline content. The most common motifs in the amino acid sequence of collagen are glycine–proline-X and glycine-X-hydroxyproline, where X is any amino acid other than glycine, proline or hydroxyproline. The average amino acid composition for fish and mammal skin is given.^[16]

Amino acid	Abundance in mammal skin (residues/1000)	Abundance in fish skin (residues/1000)
Glycine	329	339
Proline	126	108
Alanine	109	114
Hydroxyproline	95	67
Glutamic acid	74	76
Arginine	49	52
Aspartic acid	47	47
Serine	36	46
Lysine	29	26
Leucine	24	23
Valine	22	21
Threonine	19	26
Phenylalanine	13	14
Isoleucine	11	11
Hydroxylysine	6	8
Methionine	6	13
Histidine	5	7
Tyrosine	3	3
Cysteine	1	1
Tryptophan	0	0

Synthesis

First, a three-dimensional stranded structure is assembled, with the amino acids glycine and proline as its principal components. This is not yet collagen but its precursor, procollagen. Procollagen is then modified by the addition of hydroxyl groups to the amino acids proline and lysine. This step is important for later glycosylation and the formation of the triple helix structure of collagen. The hydroxylase enzymes that perform these reactions require Vitamin C as a cofactor, and a deficiency in this vitamin results in impaired collagen synthesis and the resulting disease scurvy^[17] These hydroxylation reactions are catalyzed by two different enzymes: prolyl-4-hydroxylase^[18] and lysyl-hydroxylase. Vitamin C also serves with them in inducing these reactions. In this service, one molecule of vitamin C is destroyed for each H replaced by OH. ^[19] The synthesis of collagen occurs inside and outside of the cell. The formation of collagen which results in fibrillary collagen (most common form) is discussed here. Meshwork collagen, which is often involved in the formation of filtration systems, is the other form of collagen. All types of collagens are triple helices, and the differences lie in the make-up of the alpha peptides created in step 2.

Transcription of mRNA: About 34 genes are associated with collagen formation, each coding for a specific mRNA sequence, and typically have the “COL” prefix. The beginning of collagen synthesis begins with turning on genes which are associated with the formation of a particular alpha peptide (typically alpha 1, 2 or 3).
Pre-pro-peptide formation: Once the final mRNA exits from the cell nucleus and enters into the cytoplasm, it links with the ribosomal subunits and the process of translation occurs. The early/first part of the new peptide is known as the signal sequence. The signal sequence on the N-terminal of the peptide is recognized by a signal recognition particle on the endoplasmic reticulum, which will be responsible for directing the pre-pro-peptide into the endoplasmic reticulum. Therefore, once the synthesis of new peptide is finished, it goes directly into the endoplasmic reticulum for post-translational processing. It is now known as pre-pro-collagen.
Pre-pro-peptide to pro-collagen: Three modifications of the pre-pro-peptide occur leading to the formation of the alpha peptide:
1. The signal peptide on the N-terminal is dissolved, and the molecule is now known as propeptide (not procollagen).
2. Hydroxylation of lysines and prolines on propeptide by the enzymes ‘prolyl hydroxylase’ and ‘lysyl hydroxylase’ (to produce hydroxyproline and hydroxylysine) occurs to aid cross-linking of the alpha peptides. This enzymatic step requires vitamin C as a cofactor. In scurvy, the lack of hydroxylation of prolines and lysines causes a looser triple helix (which is formed by three alpha peptides).
3. Glycosylation occurs by adding either glucose or galactose monomers onto the hydroxyl groups that were placed onto lysines, but not on prolines.
4. Once these modifications have taken place, three of the hydroxylated and glycosylated propeptides twist into a triple helix forming procollagen. Procollagen still has unwound ends, which will be later trimmed. At this point, the procollagen is packaged into a transfer vesicle destined for the Golgi apparatus.
Golgi apparatus modification: In the Golgi apparatus, the procollagen goes through one last post-translational modification before being secreted out of the cell. In this step, oligosaccharides (not monosaccharides as in step 3) are added, and then the procollagen is packaged into a secretory vesicle destined for the extracellular space.
Formation of tropocollagen: Once outside the cell, membrane bound enzymes known as ‘collagen peptidases’, remove the “loose ends” of the procollagen molecule. What is left is known as tropocollagen. Defects in this step produce one of the many collagenopathies known as Ehlers-Danlos syndrome. This step is absent when synthesizing type III, a type of fibrilar collagen.
Formation of the collagen fibril: ‘Lysyl oxidase’, an extracellular enzyme, produces the final step in the collagen synthesis pathway. This enzyme acts on lysines and hydroxylysines producing aldehyde groups, which will eventually undergo covalent bonding between tropocollagen molecules. This polymer of tropocollogen is known as a collagen fibril.

Action of lysyl oxidase

Amino acids

Collagen has an unusual amino acid composition and sequence:

Glycine is found at almost every third residue.
Proline makes up about 17% of collagen.
Collagen contains two uncommon derivative amino acids not directly inserted during translation. These amino acids are found at specific locations relative to glycine and are modified post-translationally by different enzymes, both of which require vitamin C as acofactor.
- Hydroxyproline derived from proline
- Hydroxylysine derived from lysine – depending on the type of collagen, varying numbers of hydroxylysines are glycosylated (mostly having disaccharides attached).

Cortisol stimulates degradation of (skin) collagen into amino acids.^[20]

Collagen I formation

Most collagen forms in a similar manner, but the following process is typical for type I:

Inside the cell
1. Two types of alpha chains are formed during translation on ribosomes along the rough endoplasmic reticulum (RER): alpha-1 and alpha-2 chains. These peptide chains (known as preprocollagen) have registration peptides on each end and a signal peptide.
2. Polypeptide chains are released into the lumen of the RER.
3. Signal peptides are cleaved inside the RER and the chains are now known as pro-alpha chains.
4. Hydroxylation of lysine and proline amino acids occurs inside the lumen. This process is dependent on ascorbic acid (vitamin C) as a cofactor.
5. Glycosylation of specific hydroxylysine residues occurs.
6. Triple alpha helical structure is formed inside the endoplasmic reticulum from two alpha-1 chains and one alpha-2 chain.
7. Procollagen is shipped to the Golgi apparatus, where it is packaged and secreted by exocytosis.
Outside the cell
1. Registration peptides are cleaved and tropocollagen is formed by procollagen peptidase.
2. Multiple tropocollagen molecules form collagen fibrils, via covalent cross-linking (aldol reaction) by lysyl oxidase which links hydroxylysine and lysine residues. Multiple collagen fibrils form into collagen fibers.
3. Collagen may be attached to cell membranes via several types of protein, including fibronectin and integrin.

Synthetic pathogenesis

Vitamin C deficiency causes scurvy, a serious and painful disease in which defective collagen prevents the formation of strong connective tissue. Gums deteriorate and bleed, with loss of teeth; skin discolors, and wounds do not heal. Prior to the 18th century, this condition was notorious among long-duration military, particularly naval, expeditions during which participants were deprived of foods containing vitamin C.

An autoimmune disease such as lupus erythematosus or rheumatoid arthritis^[21] may attack healthy collagen fibers.

Many bacteria and viruses secrete virulence factors, such as the enzyme collagenase, which destroys collagen or interferes with its production.

Molecular structure

A single collagen molecule, tropocollagen, is used to make up larger collagen aggregates, such as fibrils. It is approximately 300 nm long and 1.5 nm in diameter, and it is made up of three polypeptide strands (called alpha peptides, see step 2), each of which has the conformation of a left-handed helix – this should not be confused with the right-handedalpha helix. These three left-handed helices are twisted together into a right-handed triple helix or “super helix”, a cooperative quaternary structure stabilized by many hydrogen bonds. With type I collagen and possibly all fibrillar collagens, if not all collagens, each triple-helix associates into a right-handed super-super-coil referred to as the collagen microfibril. Each microfibril is interdigitated with its neighboring microfibrils to a degree that might suggest they are individually unstable, although within collagen fibrils, they are so well ordered as to be crystalline.

Three polypeptides coil to form tropocollagen. Many tropocollagens then bind together to form a fibril, and many of these then form a fibre.

A distinctive feature of collagen is the regular arrangement ofamino acids in each of the three chains of these collagen subunits. The sequence often follows the pattern Gly–Pro-X or Gly-X-Hyp, where X may be any of various other amino acid residues.^[16] Proline or hydroxyproline constitute about 1/6 of the total sequence. With glycine accounting for the 1/3 of the sequence, this means approximately half of the collagen sequence is not glycine, proline or hydroxyproline, a fact often missed due to the distraction of the unusual GX₁X₂ character of collagen alpha-peptides. The high glycine content of collagen is important with respect to stabilization of the collagen helix as this allows the very close association of the collagen fibers within the molecule, facilitating hydrogen bonding and the formation of intermolecular cross-links.^[16]This kind of regular repetition and high glycine content is found in only a few other fibrous proteins, such as silk fibroin.

Collagen is not only a structural protein. Due to its key role in the determination of cell phenotype, cell adhesion, tissue regulation and infrastructure, many sections of its non-proline-rich regions have cell or matrix association / regulation roles. The relatively high content of proline and hydroxyproline rings, with their geometrically constrained carboxyl and (secondary) amino groups, along with the rich abundance of glycine, accounts for the tendency of the individual polypeptide strands to form left-handed helices spontaneously, without any intrachain hydrogen bonding.

Because glycine is the smallest amino acid with no side chain, it plays a unique role in fibrous structural proteins. In collagen, Gly is required at every third position because the assembly of the triple helix puts this residue at the interior (axis) of the helix, where there is no space for a larger side group than glycine’s single hydrogen atom. For the same reason, the rings of the Pro and Hyp must point outward. These two amino acids help stabilize the triple helix—Hyp even more so than Pro; a lower concentration of them is required in animals such as fish, whose body temperatures are lower than most warm-blooded animals. Lower proline and hydroxyproline contents are characteristic of cold-water, but not warm-water fish; the latter tend to have similar proline and hydroxyproline contents to mammals.^[16] The lower proline and hydroxproline contents of cold-water fish and other poikilotherm animals leads to their collagen having a lower thermal stability than mammalian collagen.^[16] This lower thermal stability means that gelatin derived from fish collagen is not suitable for many food and industrial applications.

The tropocollagen subunits spontaneously self-assemble, with regularly staggered ends, into even larger arrays in the extracellular spaces of tissues.^[22]^[23] Additional assembly of fibrils is guided by fibroblasts, which deposit fully formed fibrils from fibripositors.^[2] In the fibrillar collagens, the molecules are staggered from each other by about 67 nm (a unit that is referred to as ‘D’ and changes depending upon the hydration state of the aggregate). Each D-period contains four plus a fraction collagen molecules, because 300 nm divided by 67 nm does not give an integer (the length of the collagen molecule divided by the stagger distance D). Therefore, in each D-period repeat of the microfibril, there is a part containing five molecules in cross-section, called the “overlap”, and a part containing only four molecules, called the “gap”.^[24] The triple-helices are also arranged in a hexagonal or quasihexagonal array in cross-section, in both the gap and overlap regions.^[24]^[25]

There is some covalent crosslinking within the triple helices, and a variable amount of covalent crosslinking between tropocollagen helices forming well organized aggregates (such as fibrils).^[26] Larger fibrillar bundles are formed with the aid of several different classes of proteins (including different collagen types), glycoproteins and proteoglycans to form the different types of mature tissues from alternate combinations of the same key players.^[23] Collagen’s insolubility was a barrier to the study of monomeric collagen until it was found that tropocollagen from young animals can be extracted because it is not yet fully crosslinked. However, advances in microscopy techniques (i.e. electron microscopy (EM) and atomic force microscopy (AFM)) and X-ray diffraction have enabled researchers to obtain increasingly detailed images of collagen structure in situ. These later advances are particularly important to better understanding the way in which collagen structure affects cell–cell and cell–matrix communication, and how tissues are constructed in growth and repair, and changed in development and disease.^[27]^[28] For example, using AFM–based nanoindentation it has been shown that a single collagen fibril is a heterogeneous material along its axial direction with significantly different mechanical properties in its gap and overlap regions, correlating with its different molecular organizations in these two regions.^[29]

Collagen fibrils/aggregates are arranged in different combinations and concentrations in various tissues to provide varying tissue properties. In bone, entire collagen triple helices lie in a parallel, staggered array. 40 nm gaps between the ends of the tropocollagen subunits (approximately equal to the gap region) probably serve as nucleation sites for the deposition of long, hard, fine crystals of the mineral component, which is (approximately) Ca₁₀(OH)₂(PO₄)₆.^[30] Type I collagen gives bone its tensile strength.

Associated disorders

Collagen-related diseases most commonly arise from genetic defects or nutritional deficiencies that affect the biosynthesis, assembly, postranslational modification, secretion, or other processes involved in normal collagen production.

**Genetic Defects of Collagen Genes**
Type	Notes	Gene(s)	Disorders
I	This is the most abundant collagen of the human body. It is present in scar tissue, the end product when tissue heals by repair. It is found in tendons, skin, artery walls, cornea, the endomysiumsurrounding muscle fibers, fibrocartilage, and the organic part of bones and teeth.	COL1A1, COL1A2	Osteogenesis imperfecta, Ehlers–Danlos syndrome, Infantile cortical hyperostosis a.k.a. Caffey’s disease
II	Hyaline cartilage, makes up 50% of all cartilage protein. Vitreous humour of the eye.	COL2A1	Collagenopathy, types II and XI
III	This is the collagen of granulation tissue, and is produced quickly by young fibroblasts before the tougher type I collagen is synthesized. Reticular fiber. Also found in artery walls, skin, intestines and the uterus	COL3A1	Ehlers–Danlos syndrome, Dupuytren’s contracture
IV	Basal lamina; eye lens. Also serves as part of the filtration system in capillaries and the glomeruli ofnephron in the kidney.	COL4A1, COL4A2,COL4A3, COL4A4,COL4A5, COL4A6	Alport syndrome, Goodpasture’s syndrome
V	Most interstitial tissue, assoc. with type I, associated with placenta	COL5A1, COL5A2,COL5A3	Ehlers–Danlos syndrome (Classical)
VI	Most interstitial tissue, assoc. with type I	COL6A1, COL6A2,COL6A3, COL6A5	Ulrich myopathy, Bethlem myopathy,Atopic dermatitis^[31]
VII	Forms anchoring fibrils in dermoepidermal junctions	COL7A1	Epidermolysis bullosa dystrophica
VIII	Some endothelial cells	COL8A1, COL8A2	Posterior polymorphous corneal dystrophy 2
IX	FACIT collagen, cartilage, assoc. with type II and XI fibrils	COL9A1, COL9A2,COL9A3	EDM2 and EDM3
X	Hypertrophic and mineralizing cartilage	COL10A1	Schmid metaphyseal dysplasia
XI	Cartilage	COL11A1, COL11A2	Collagenopathy, types II and XI
XII	FACIT collagen, interacts with type I containing fibrils, decorin and glycosaminoglycans	COL12A1	–
XIII	Transmembrane collagen, interacts with integrin a1b1, fibronectin and components of basement membranes like nidogen and perlecan.	COL13A1	–
XIV	FACIT collagen, also known as undulin	COL14A1	–
XV	–	COL15A1	–
XVI	–	COL16A1	–
XVII	Transmembrane collagen, also known as BP180, a 180 kDa protein	COL17A1	Bullous pemphigoid and certain forms of junctional epidermolysis bullosa
XVIII	Source of endostatin	COL18A1	–
XIX	FACIT collagen	COL19A1	–
XX	–	COL20A1	–
XXI	FACIT collagen	COL21A1	–
XXII	–	COL22A1	–
XXIII	MACIT collagen	COL23A1	–
XXIV	–	COL24A1	–
XXV	–	COL25A1	–
XXVI	–	EMID2	–
XXVII	–	COL27A1	–
XXVIII	–	COL28A1	–

In addition to the above-mentioned disorders, excessive deposition of collagen occurs in scleroderma.

Diseases

One thousand mutations have been identified in twelve out of more than twenty types of collagen. These mutations can lead to various diseases at the tissue level.^[32]

Osteogenesis imperfecta – Caused by a mutation in type 1 collagen, dominant autosomal disorder, results in weak bones and irregular connective tissue, some cases can be mild while others can be lethal, mild cases have lowered levels of collagen type 1 while severe cases have structural defects in collagen.^[33]

Chondrodysplasias – Skeletal disorder believed to be caused by a mutation in type 2 collagen, further research is being conducted to confirm this.^[34]

Ehlers-Danlos Syndrome – Six different types of this disorder, which lead to deformities in connective tissue. Some types can be lethal, leading to the rupture of arteries. Each syndrome is caused by a different mutation, for example type four of this disorder is caused by a mutation in collagen type 3.^[35]

Alport syndrome – Can be passed on genetically, usually as X-linked dominant, but also as both an autosomal dominant and autosomal recessive disorder, sufferers have problems with their kidneys and eyes, loss of hearing can also develop in during the childhood or adolescent years.^[36]

Osteoporosis – Not inherited genetically, brought on with age, associated with reduced levels of collagen in the skin and bones, growth hormone injections are being researched as a possible treatment to counteract any loss of collagen.^[37]

Knobloch syndrome – Caused by a mutation in the COL18A1 gene that codes for the production of collagen XVIII. Patients present with protrusion of the brain tissue and degeneration of the retina, an individual who has family members suffering from the disorder are at an increased risk of developing it themselves as there is a hereditary link.^[32]

Characteristics

Collagen is one of the long, fibrous structural proteins whose functions are quite different from those of globular proteins, such as enzymes. Tough bundles of collagen calledcollagen fibers are a major component of the extracellular matrix that supports most tissues and gives cells structure from the outside, but collagen is also found inside certain cells. Collagen has great tensile strength, and is the main component of fascia, cartilage, ligaments, tendons, bone and skin.^[38]^[39] Along with elastin and soft keratin, it is responsible for skin strength and elasticity, and its degradation leads to wrinkles that accompany aging.^[10] It strengthens blood vessels and plays a role in tissue development. It is present in the cornea and lens of the eye in crystalline form. It may be one of the most abundant proteins in the fossil record, given that it appears to fossilize frequently, even in bones from the Mesozoic and Paleozoic.^[40]

Uses

Collagen has a wide variety of applications, from food to medical. For instance, it is used in cosmetic surgery and burn surgery. It is widely used in the form of collagen casings for sausages, which are also used in the manufacture of musical strings.

If collagen is subject to sufficient denaturation, e.g. by heating, the three tropocollagen strands separate partially or completely into globular domains, containing a different secondary structure to the normal collagen polyproline II (PPII), e.g. random coils. This process describes the formation of gelatin, which is used in many foods, including flavoredgelatin desserts. Besides food, gelatin has been used in pharmaceutical, cosmetic, and photography industries.^[41] From a nutritional point of view, collagen and gelatin are a poor-quality sole source of protein since they do not contain all the essential amino acids in the proportions that the human body requires—they are not ‘complete proteins‘ (as defined by food science, not that they are partially structured). Manufacturers of collagen-based dietary supplements usually containing hydrolyzed collagen claim that their products can improve skin and fingernail quality as well as joint health. However, mainstream scientific research has not shown strong evidence to support these claims.^[42]Individuals with problems in these areas are more likely to be suffering from some other underlying condition (such as normal aging, dry skin, arthritis etc.) rather than just a protein deficiency.

From the Greek for glue, kolla, the word collagen means “glue producer” and refers to the early process of boiling the skin and sinews of horses and other animals to obtain glue. Collagen adhesive was used by Egyptians about 4,000 years ago, and Native Americans used it in bows about 1,500 years ago. The oldest glue in the world, carbon-dated as more than 8,000 years old, was found to be collagen—used as a protective lining on rope baskets and embroidered fabrics, and to hold utensils together; also in crisscross decorations on human skulls.^[43] Collagen normally converts to gelatin, but survived due to dry conditions. Animal glues are thermoplastic, softening again upon reheating, and so they are still used in making musical instruments such as fine violins and guitars, which may have to be reopened for repairs—an application incompatible with tough, synthetic plastic adhesives, which are permanent. Animal sinews and skins, including leather, have been used to make useful articles for millennia.

Gelatin-resorcinol–formaldehyde glue (and with formaldehyde replaced by less-toxic pentanedial and ethanedial) has been used to repair experimental incisions in rabbit lungs.^[44]

History

The molecular and packing structures of collagen have eluded scientists over decades of research. The first evidence that it possesses a regular structure at the molecular level was presented in the mid-1930s.^[45]^[46] Since that time, many prominent scholars, including Nobel laureates Crick, Pauling, Rich and Yonath, and others, including Brodsky,Berman, and Ramachandran, concentrated on the conformation of the collagen monomer. Several competing models, although correctly dealing with the conformation of each individual peptide chain, gave way to the triple-helical “Madras” model of Ramachandran, which provided an essentially correct model of the molecule’s quaternary structure^[47]^[48]^[49] although this model still required some refinement.^[50] ^{[clarification needed]} ^[51]^[52]^[53]^[54] The packing structure of collagen has not been defined to the same degree outside of the fibrillar collagen types, although it has been long known to be hexagonal or quasi-hexagonal.^[25]^[55]^[56] As with its monomeric structure, several conflicting models alleged that either the packing arrangement of collagen molecules is ‘sheet-like’ or microfibrillar.^[50]^[57]^[58] The microfibrillar structure of collagen fibrils in tendon, cornea and cartilage has been directly imaged by electron microscopy.^[59]^[60]^[61] The microfibrillar structure of tail tendon, as described by Fraser, Miller, and Wess (amongst others), was modeled as being closest to the observed structure,^[50] although it oversimplified the topological progression of neighboring collagen molecules, and hence did not predict the correct conformation of the discontinuous D-periodic pentameric arrangement termed simply: the microfibril.^[24]^[62] Various cross linking agents like L-Dopaquinone, embeline, potassium embelate and 5-O-methyl embelin could be developed as potential cross-linking/stabilization agents of collagen preparation and its application as wound dressing sheet in clinical applications is enhanced.^[63]

References

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External links

12 types of collagen

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Illegal Drugs in Dietary Supplements

drugs Comments Off

May 132016

Illegal Drugs in Dietary Supplements

Forbidden stimulant oxilofrine found in various dietary supplements sold in the USA

http://www.chemistryviews.org/details/news/9278771/Illegal_Drugs_in_Dietary_Supplements.html

Pharmaceutical doses of the banned stimulant oxilofrine found in dietary supplements sold in the USA,
Pieter A. Cohen, Bharathi Avula, Bastiaan Venhuis, John C. Travis, Yan-Hong Wang, Ikhlas A. Khan,
Drug Test. Anal. 2016.
DOI: 10.1002/dta.1976

Oxilofrine (also known as methylsynephrine, hydroxyephrine, oxyephrine, and 4-HMP) is a stimulant drug^[1] and is anamphetamine chemically related to ephedrine and to synephrine.

Oxilofrine is currently a WADA prohibited substance when used in competition.^[2] It is an ingredient found in some dietary supplements.

Publicized cases

In 2009, Brazilian/American cyclist Flávia Oliveira was suspended for 2 years after taking a supplement known as “HyperDrive 3.0+” which contained methylsynephrine, a chemical equivalent of Oxilofrine, among other substances. ^[3] Her sentence was eventually reduced to 18 months after an appeal as there was enough evidence that she had unknowingly consumed said substance as the old label did not list methylsynephrine.^[4]
On July 14, 2013, Jamaican runners Asafa Powell and Sherone Simpson tested positive for Oxilofrine prior to the 2013 World Athletics Championships. ^[5] Powell, however, maintained that he did not take any banned supplements knowingly or willfully.^[6]Powell voluntarily withdrew as a result of the test. On 10 April 2014, both athletes received an 18-month suspension from competing, which was set to expire in December that year.^[7] However, after appealing to the Court of Arbitration for Sport (CAS), both athletes’ suspensions were lifted on 14 July 2014.^[8]
On July 16, 2015, Boston Red Sox pitching prospect Michael Kopech was suspended without pay for 50 games after testing positive for Oxilofrine, which is a banned substance under the Minor League Drug Prevention and Treatment Program. Kopech denied knowingly taking the substance.^[9]

References

Fourcroy, Jean L. (2008). Pharmacology, doping and sports: a scientific guide for athletes, coaches, physicians, scientists and administrators. Taylor & Francis. ISBN 978-0-415-42845-3.
http://list.wada-ama.org/prohibited-in-competition/prohibited-substances/
Charles Pelkey (2010-04-13). “Oliveira suspended for two years”. Velonews.
Charles Pelkey (2011-02-24). “Court of Arbitration for Sport reduces Flavia Oliveira suspension”. Velonews.
Reuters. “Jamaicans Powell, Simpson test positive – SuperSport – Athletics”. SuperSport. Retrieved 2013-07-15.
“Jamaican Sprinter Asafa Powell slapped 18-month ban for doping”. IANS. news.biharprabha.com. Retrieved 10 April 2014.
“Asafa Powell banned for 18 months for doping”. BBC Sport. 10 April 2014. Archived from the original on 9 May 2014.
Drayton, John (14 July 2014). “Asafa Powell and Sherone Simpson given green light to return to action after sprinters have doping bans reduced to six months”. Mail Online. Retrieved14 July 2014.
Danny Wild (16 July 2015). “Red Sox No. 10 prospect Kopech suspended”. MiLB.com. Retrieved 8 March 2016

Oxilofrine
Systematic (IUPAC) name

(1S,2R)-(±)-4-(1-Hydroxy-2-methylamino-propyl)phenol
Legal status
Legal status	Uncontrolled
Identifiers
CAS Number	365-26-4
ATC code	none
PubChem	CID 9701
ChemSpider	9320
UNII	F49638UBDR
KEGG	D08314
ChEMBL	CHEMBL30400
Chemical data
Formula	C₁₀H₁₅NO₂
Molar mass	181.23 g/mol