AUTHOR OF THIS BLOG

DR ANTHONY MELVIN CRASTO, WORLDDRUGTRACKER
Feb 092017
 

STR1

 

(3R)-4-[2-chloro-6-[[(R)-methylsulfinyl]methyl]pyrimidin-4-yl]-3-methyl-morpholine

STR1 STR2

Synthesis of (3R)-4-[2-chloro-6-[[(R)-methylsulfinyl]methyl]pyrimidin-4-yl]-3-methyl-morpholine (10)

off-white solid (53.9 kg, 68.3% yield). 1H NMR (400 MHz, DMSO-d6, δ): 1.20 (d, J = 6.8 Hz, 3 H), 2.52 (m, 1 H), 2.63 (s, 3 H), 3.21 (m, 1 H), 3.44 (m, 1 H), 3.58 (dd, J = 11.6, 3.1 Hz, 1 H), 3.72 (d, J = 11.5 Hz, 1 H), 3.92 (m, 3 H), 4.07 (d, J = 12.4 Hz, 1 H), 6.80 (s, 1 H); Assay (HPLC) 99%; Assay (QNMR) 100%; Chiral purity (HPLC) (R,R)-diastereoisomer 99.6%, (R,S)-diastereoisomer 0.4%.

 

Abstract Image

A Baeyer–Villiger monooxygenase enzyme has been used to manufacture a chiral sulfoxide drug intermediate on a kilogram scale. This paper describes the evolution of the biocatalytic manufacturing process from the initial enzyme screen, development of a kilo lab process, to further optimization for plant scale manufacture. Efficient gas–liquid mass transfer of oxygen is key to obtaining a high yield.

Development and Scale-up of a Biocatalytic Process To Form a Chiral Sulfoxide

The Departments of Pharmaceutical Sciences and Pharmaceutical Technology and Development, AstraZeneca, Silk Road Business Park, Macclesfield, Cheshire SK10 2NA, United Kingdom
Org. Process Res. Dev., Article ASAP
DOI: 10.1021/acs.oprd.6b00391
Publication Date (Web): January 4, 2017
Copyright © 2017 American Chemical Society
*Tel: +44 (0)1625-519149. E-mail: william.goundry@astrazeneca.com.
Figure
Examples of biologically active molecules containing a sulfoxide or sulfoximine: esomeprazole (3), aprikalim (4), oxisurane (5), OPC-29030 (6), ZD3638 (7), buthionine sulfoximine (8), and AZD6738 (9).

“ALL FOR DRUGS” CATERS TO EDUCATION GLOBALLY, No commercial exploits are done or advertisements added by me. This article is a compilation for educational purposes only.

P.S. : The views expressed are my personal and in no-way suggest the views of the professional body or the company that I represent

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Feb 092017
 

STR1 STR2 STR3

(±)-trans-ethyl 2-(3,4-difluorophenyl)Cyclopropanecarboxylate

C12H12F2O2

GC-MS (EI) m/z: [M]+ calc. for C12H12F2O2 + : 226.08; found: 226.08.

δH (400 MHz, CDCl3): 1.25 (1H, ddd, 3 J 8.4 Hz, 3 J 6.4 Hz, 2 J 4.5 Hz , 3-H); 1.28 (3H, t 3 J 6.4 Hz CH3Ethyl) 1.57-1.62 (2H, m, 3 J 9.2 Hz, 3 J 5.2 Hz, 2 J 4.5 Hz, 3-H + H2O), 1.84 (1H, ddd, 3 J 8.5 Hz, 3 J 5.3 Hz, 3 J 4.3 Hz , 2-H), 2.47 (1H, ddd, 3 J 9.5 Hz, 3 J 6.4 Hz, 3 J 4.2 Hz , 1-H), 4.17 (2H, q, 3 J 6.3 Hz, CH2Ethyl) 6.81-6.87 (1H, m, 3 J 8.5 Hz, 4 J 7.6 Hz, 4 J 2.4 Hz, 6-H’ ), 6.88 (1H, ddd, 3 J 11.5 Hz, 4 J 7.6 Hz, 4 J 2.2 Hz, 2-H’) 7.06 (1H, dt, 3 J 10.3 Hz, 3 J 8.2 Hz. 5-H’).

δc (400 MHz, CDCl3): 14.27 (CH3Ethyl), 16.84 (3-C) 24.04 (1-C), 25.14 (d, 4 J 1.4, 2-C), 60.71 (CH2Ethyl), 114.74 (d, 2 J 19 Hz, 2-C’), 117.09 (d, 2 J 18 Hz, 5-C’), 122.25 (dd, 3 J 6.1 Hz, 4 J 3.4 Hz, 6- C’), 137.06 (dd, 3 J 6.1 Hz, 4 J 3.4 Hz, 1- C’), 149.2 (dd, 1 J 248 Hz, 2 J 13 Hz, 4-C’) 151.32 (dd, 1 J 249 Hz, 2 J 12.5 Hz, 3-C’) 172.87 (Ccarbonyl).

[ ] 20 a D = -381.9 (c 1.0 in EtOH) for (1R,2R)-3, ee = 95%

Abstract Image

In this study a batch reactor process is compared to a flow chemistry approach for lipase-catalyzed resolution of the cyclopropanecarboxylate ester (±)-3. (1R,2R)-3 is a precursor of the amine (1R,2S)-2 which is a key building block of the API ticagrelor. For both flow and batch operation, the biocatalyst could be recycled several times, whereas in the case of the flow process the reaction time was significantly reduced.

Comparison of a Batch and Flow Approach for the Lipase-Catalyzed Resolution of a Cyclopropanecarboxylate Ester, A Key Building Block for the Synthesis of Ticagrelor

School of Chemistry, University of Manchester, Manchester Institute of Biotechnology, 131 Princess Street, Manchester M1 7DN, United Kingdom
Chemessentia, SRL – Via G. Bovio, 6-28100 Novara, Italy
§ Institute of Process Research and Development, School of Chemistry, University of Leeds, Woodhouse Lane, Leeds, LS2 9JT, United Kingdom
Org. Process Res. Dev., Article ASAP
DOI: 10.1021/acs.oprd.6b00346
Publication Date (Web): December 22, 2016
Copyright © 2016 American Chemical Society

“ALL FOR DRUGS” CATERS TO EDUCATION GLOBALLY, No commercial exploits are done or advertisements added by me. This article is a compilation for educational purposes only.

P.S. : The views expressed are my personal and in no-way suggest the views of the professional body or the company that I represent

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Feb 012017
 

 

STR1

2,2′-(1-(tert-Butoxycarbonyl)pyrrolidine-3,4-diyl)diacetic Acid

STR1 STR2 STR3 str4 str5

2,2′-(1-(tert-Butoxycarbonyl)pyrrolidine-3,4-diyl)diacetic Acid 

as a white solid. Mp: 162–163 °C, % purity: 94.09% (HPLC);
1H NMR (DMSO-d6, 400 MHz) δ: 1.38 (s, 9H), 2.10–2.18 (m, 2H), 2.28–2.32 (m, 2H), 2.49–2.50 (m, 2H, merged with DMSO peak), 2.97–3.03 (m, 2H), 3.33–3.40 (m, 2H), 12.23 (bs, 2H); 1H NMR (CD3OD, 400 MHz) δ: 1.46 (s, 9H), 2.26 (ddd, J1 = 2.8 Hz, J2 = 9.2 Hz, J3 = 16.0 Hz, 2H), 2.43 (dd, J1 = 5.2 Hz, J2 = 16.0 Hz, 2H), 2.69 (m, 2H), 3.16 (dd, J1 = 5.2 Hz, J2 = 10.8 Hz, 2H), 3.49–3.54 (m, 2H);
13C NMR (DMSO-d6, 100 MHz) δ: 28.49, 32.97, 36.49, 37.31, 50.10, 50.20, 78.67, 154.05, 173.96;
IR (KBr): ν = 871, 933, 1143, 1166, 1292, 1411, 1689, 1708, 2881, 2929, 2980, 3001 cm–1;
TOFMS: [C13H21NO6 – H+]: calculated 286.1296, found 286.1031(100%).
HPLC conditions were as follows for compound ; Agilent 1100 series, column: YMC J’SPHERE C18 (150 mm X 4.6 mm) 4µm with mobile phases A (0.05% TFA in water) and B (acetonitrile). Detection was at 210 nm, flow was set at 1.0 mL/min, and the temperature was 30 °C (Run time: 45 min). Gradient: 0 min, A = 90%, B = 10%; 5.0 min, A = 90%, B = 10%; 25 min, A = 0%, B = 100%; 30 min, A = 0%, B = 100%, 35 min, A = 90%, B = 10%; 45 min, A = 90%, B = 10%.
Org. Process Res. Dev., Article ASAP
DOI: 10.1021/acs.oprd.6b00399
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Jan 312017
 

str5

Dimethyl 4,4′-(Benzylazanediyl)(2E,2′E)-bis(but-2-enoate)

STR1

IR (CHCl3): ν = 758, 1215, 1278, 1437, 1660, 1720, 2806, 2953, 3020, 3421 cm–1;

 

STR2

13C NMR (CDCl3, 100 MHz) δ: 51.53, 53.42, 58.37, 122.66, 127.28, 128.41, 128.55, 128.76, 138.24, 145.84, 166.58;

 

STR3

1H NMR (CDCl3, 400 MHz) δ: 3.23 (dd, J1 = 1.6 Hz, J2 = 6.0 Hz, 4H), 3.62 (s, 2H), 3.75 (s, 6H), 6.07 (dt, J1 = 1.6 Hz, J2 = 16.0 Hz, 2H), 6.97 (dt, J1 = 6.0 Hz, J2 = 16.0 Hz, 2H), 7.25–7.34 (m, 5H-merged with CDCl3 proton);

 

str4

TOFMS: [C17H21NO4 + H+]: calculated 304.1543, found 304.1703(100%).

str5

 

UPLC conditions were as follows for compound 11; Acquity Waters, column: BEH C18 (2.1 mm X 100 mm) 1.7 µm with mobile phases A (0.05% TFA in water) and B (acetonitrile). Detection was at 220 nm, flow was set at 0.4 mL/min, and the temperature was 30 °C (Run time: 9 min). Gradient: 0 min, A = 90%, B = 10%; 0.5 min, A = 90%, B = 10%; 6.0 min, A = 0%, B = 100%; 7.5 min, A = 0%, B = 100%; 7.6 min, A = 90%, B = 10%; 9.0 min, A = 90%, B = 10%.

 

Dimethyl 4,4′-(Benzylazanediyl)(2E,2′E)-bis(but-2-enoate) (11)

as a yellow oil. % purity: 93.4% (UPLC);
1H NMR (CDCl3, 400 MHz) δ: 3.23 (dd, J1 = 1.6 Hz, J2 = 6.0 Hz, 4H), 3.62 (s, 2H), 3.75 (s, 6H), 6.07 (dt, J1 = 1.6 Hz, J2 = 16.0 Hz, 2H), 6.97 (dt, J1 = 6.0 Hz, J2 = 16.0 Hz, 2H), 7.25–7.34 (m, 5H-merged with CDCl3 proton);
13C NMR (CDCl3, 100 MHz) δ: 51.53, 53.42, 58.37, 122.66, 127.28, 128.41, 128.55, 128.76, 138.24, 145.84, 166.58;
IR (CHCl3): ν = 758, 1215, 1278, 1437, 1660, 1720, 2806, 2953, 3020, 3421 cm–1;
TOFMS: [C17H21NO4 + H+]: calculated 304.1543, found 304.1703(100%).
Org. Process Res. Dev., Article ASAP
DOI: 10.1021/acs.oprd.6b00399
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1-Bromo-4-fluoro-2-((2-iodobenzyl)oxy)benzene

 Uncategorized  Comments Off on 1-Bromo-4-fluoro-2-((2-iodobenzyl)oxy)benzene
Jan 252017
 

STR1

1-Bromo-4-fluoro-2-((2-iodobenzyl)oxy)benzene

CAS 1161931-51-6

STR1 STR2

Mp 89.8–92.3 °C.

IR (neat, ATR): 3072 (w), 1482 (s), 1451 (s), 1294 (s), 1294 (s) cm–1.

1H NMR (399 MHz, DMSO-d6) δ 5.12 (s, 2H), 6.81 (td, J = 8.49, 2.77 Hz, 1H), 7.14 (td, J = 7.64, 1.65 Hz, 1H), 7.18 (dd, J = 10.90, 2.82 Hz, 1H), 7.46 (td, J = 7.52, 0.92 Hz, 1H), 7.60 (dd, J = 7.64, 1.41 Hz, 1H), 7.62 (dd, J = 8.66, 6.23 Hz, 1H), 7.92 (dd, J = 7.83, 0.83 Hz, 1H).

13C NMR (100 MHz, DMSO-d6) δ 74.5, 99.2, 102.4 (d, J = 27.1 Hz), 105.8 (d, J = 3.4 Hz), 108.9 (d, J = 22.5 Hz), 128.5, 129.8, 130.3, 133.6 (d, J = 9.9 Hz), 138.0, 139.2, 155.4 (d, J = 10.7 Hz), 162.2 (d, J = 244.3 Hz).

GCMS: m/z [M]+ calcd for C13H9BrFIO: 405.88600; found: 405.88620.

1H AND 13C NMR PREDICT

STR1 STR2 STR3 str4

 

Org. Process Res. Dev., Article ASAP

“ALL FOR DRUGS” CATERS TO EDUCATION GLOBALLY, No commercial exploits are done or advertisements added by me. This article is a compilation for educational purposes only.

P.S. : The views expressed are my personal and in no-way suggest the views of the professional body or the company that I represent

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Brc2ccc(F)cc2OCc1ccccc1I
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Synthesis of (E)-2,4-Dinitro-N-((2E,4E)-4-phenyl-5-(pyrrolidin-1-yl)penta-2,4-dienylidene)aniline

 spectroscopy, SYNTHESIS, Uncategorized  Comments Off on Synthesis of (E)-2,4-Dinitro-N-((2E,4E)-4-phenyl-5-(pyrrolidin-1-yl)penta-2,4-dienylidene)aniline
Dec 212016
 

str1

Cas 1204588-48-6
MF C21 H20 N4 O4
MW 392.41
Benzenamine, 2,​4-​dinitro-​N-​[(2E,​4E)​-​4-​phenyl-​5-​(1-​pyrrolidinyl)​-​2,​4-​pentadien-​1-​ylidene]​-​, [N(E)​]​-
(E)-2,4-Dinitro-N-((2E,4E)-4-phenyl-5-(pyrrolidin-1-yl)penta-2,4-dienylidene)aniline
str1

 

 

Molbank 2009, 2009(3), M604; doi:10.3390/M604

Synthesis of (E)-2,4-Dinitro-N-((2E,4E)-4-phenyl-5-(pyrrolidin-1-yl)penta-2,4-dienylidene)aniline
Nosratollah Mahmoodi 1,*, Manuchehr Mamaghani 1, Ali Ghanadzadeh 2, Majid Arvand 3 and Mostafa Fesanghari 1
1Laboratory of Organic Chemistry, Faculty of Science, University of Guilan, P.O.Box 1914, Rasht, Iran,
2Departments of Physical Chemistry, Faculty of Science, University of Guilan, P.O.Box 1914, Rasht, Iran
3Departments of Analytical Chemistry, Faculty of Science, University of Guilan, P.O.Box 1914, Rasht, Iran
*Author to whom correspondence should be addressed
mahmoodi@guilan.ac.ir, m-chem41@guilan.ac.ir, aggilani@guilan.ac.ir, arvand@guilan.ac.ir, nosmahmoodi@gmail.com

Abstract:

(E)-2,4-Dinitro-N-((2E,4E)-4-phenyl-5-(pyrrolidin-1-yl)penta-2,4-dienylidene) aniline dye was prepared in one pot by reaction of premade N-2,4-dinitrophenyl-3-phenylpyridinium chloride (DNPPC) and pyrrolidine in absolute MeOH.
Keywords:

N-2,4-dinitrophenyl-3-phenylpyridinium chloride (DNPPC); photochromic; pyridinium salt

N-2,4-Dinitrophenyl-3-phenylpyridinium chloride (DNPPC) 1 was prepared according to the literature method [1,2,3,4,5,6,7]. Recently, we became interested in the synthesis of photochromic compounds [8,9,10]. The UV-Vis spectra under irradiation of UV light of dye 2 indicate photochromic properties for this molecule. The salt 1 was premade and typically isolated and purified by recrystallization and characterized. To a solution of 1-chloro-2,4-dinitrobenzene (1.42 g, 7.01 mmol) in acetone (10 mL) was added 3-phenylpyridine (1.0 mL, 6.97 mmol). The reaction was heated at reflux for 48 h. The solvent was removed under reduced pressure and the red residue was stirred in hexanes. The precipitated product was collected by vacuum filtration to afford pure pyridinium salt 1 as a reddish brown solid (2.23 g, 6.25 mmol, 90%). 1H NMR (CDCl3, 500 MHz): δ (ppm) 9.9 (s, 1H), 9.4 (d, J = 6.0 Hz, 1H), 9.3 (d, J = 8.3 Hz, 1H), 9.2 (d, J = 2.2 Hz, 1H), 9.0 (dd, J = 8.7, 2.4 Hz, 1H), 8.5-8.6 (m, 2H), 8.0 (d, J = 7.3 Hz, 2H), 7.6- 7.7 (m, 3H); 13C NMR (CDCl3, 125 MHz): δ (ppm) 149.2, 145.6, 144.3, 144.2, 143.0, 139.2, 138.7, 132.5, 132.3, 130.6, 130.2, 129.6, 128.0, 127.6, 121.3; IR (KBr pellet) 3202, 3129, 2994, 2901, 1609 cm-1; m. p. = 182-183 °C; HRMS m/z Calcd for C17H12N3O4+ (M)+ 322.0828, found 322.0836.
Molbank 2009 m604 i001
Reaction of pyrrolidine with salt (1) leads to the opening of the pyridinium ring and formation of dye 2. This dye was prepared from reaction of salt 1 (0.5 g, 1.4 mmol) in 5 mL absolute MeOH after cooling a reaction mixture to -10oC and keeping at this temperature for 15 min. To this was added pyrrolidine (0.1 g, 1.4 mmol) in 3 mL absolute MeOH over a period of 10 min. The prepared solid was filtered, washed with CH2Cl2, dried and recrystallized from n-hexane to yield 68% (0.37 g, 0.95 mmol) of pure metallic greenish-brown 2,
m.p. = 146 oC.
IR (KBr): 3040, 2950, 1616, 1514, 1492, 1469, 1321, 1215, 1170, 1105, 956, 904, 862, 727 cm-1.
1H NMR (500 MHz, CDCl3): δ (ppm) 8.7 (d, J = 2.4 Hz, 1H) 8.3 (dd, J = 2.4, 8.84 Hz, 1H), 8.0 (s, 1H), 7.5 (d, J = 7.4 Hz, 2H), 7.4-7.5 (t, J = 7.5 Hz, 2H), 7.3-7.4 (m, 1H), 7.2 (d, J = 12.5 Hz, 1H), 7.1 (d, J = 8.9 Hz, 1H), 7.0 (d, J = 12.1 Hz, 1H), 5.4 (t, J = 12.2 Hz, 1H), 3.3 (br, 4H), 2.0 (br, 4H);
13C NMR (125 MHz, CDCl3): δ (ppm) 22.0, 55.6, 114.7, 117.4, 120.0, 124.1, 126.4, 128.7, 128,8, 129.0, 132.7, 137.1, 137.3, 142.9, 147.8, 150.2, 163.8.
Anal. Calcd for C21H20N4O4: %C = 64.28, %H = 5.14, %N = 14.28. Found: %C = 64.08, %H = 5.11, %N = 14.07.

str1

 

 

1H NMR PREDICT

str0

ACTUAL….

1H NMR (500 MHz, CDCl3): δ (ppm) 8.7 (d, J = 2.4 Hz, 1H) 8.3 (dd, J = 2.4, 8.84 Hz, 1H), 8.0 (s, 1H), 7.5 (d, J = 7.4 Hz, 2H), 7.4-7.5 (t, J = 7.5 Hz, 2H), 7.3-7.4 (m, 1H), 7.2 (d, J = 12.5 Hz, 1H), 7.1 (d, J = 8.9 Hz, 1H), 7.0 (d, J = 12.1 Hz, 1H), 5.4 (t, J = 12.2 Hz, 1H), 3.3 (br, 4H), 2.0 (br, 4H);

str0

 

13 C NMR PREDICT

 

str1

ACTUAL…….13C NMR (125 MHz, CDCl3): δ (ppm) 22.0, 55.6, 114.7, 117.4, 120.0, 124.1, 126.4, 128.7, 128,8, 129.0, 132.7, 137.1, 137.3, 142.9, 147.8, 150.2, 163.8.

str3

////////////Synthesis, (E)-2,4-Dinitro-N-((2E,4E)-4-phenyl-5-(pyrrolidin-1-yl)penta-2,4-dienylidene)aniline

[O-][N+](=O)c3ccc(\N=C\C=C\C(=C/N1CCCC1)c2ccccc2)c([N+]([O-])=O)c3

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NMR, 3-[3-(benzoylamino)-4-hydroxylphenyl] propanoic acid

 spectroscopy  Comments Off on NMR, 3-[3-(benzoylamino)-4-hydroxylphenyl] propanoic acid
Jul 022015
 

 

 

http://www.google.com/patents/WO2008022462A1?cl=en

 

1 H-NMR Spectrum of Compound 35…………3-[3-(benzoylamino)-4-hydroxylphenyl] propanoic acid

1H-NMR (Acetone-D6) δ: 2.60 (t, 2H, J = 7.4, H- 3), 2.84 (t, 2H, J = 7.9, H-2), 6.89 (d, IH, J = 8.2, H-8), 7.00 (dd, IH, J = 2.1 , 8.25, H- 9), 7.57 (m, 4H, H-5, H-4′, H-5′, H-61), 8.05 (d, 2H, J = 8.2, H-3′, H-7′), 9.07 (broad s, IH, NH), 9.54 (broad s, IH, OH), 10.58 (broad s, IH, CO2H).

Figure imgf000063_0001
Figure imgf000063_0002

13C-NMR Spectrum of Compound 35

13C-NMR (Acetone- D6) δ: 30.87 (C- 3), 36.21 (C- 2), 118.69 (C- 8), 123.31 (C-5), 123.41 (C- 6), 126.88 (C- 9), 127.37 (C- 4), 128.54 (C-41, C-61), 129.61 (C-31, C-7′), 132.99 (C-51), 134.99 (C-21), 148.03 (C-7), 167.34 (C-I1), 173.94 (C-I ).

Figure imgf000064_0001
Figure imgf000064_0002

13C-NMR Spectrum of Compound 35

Figure imgf000065_0001
Figure imgf000065_0002

COSY-NMR Spectrum of Compound 35

Figure imgf000066_0001
Figure imgf000066_0002

COSY-NMR Spectrum of Compound 35

 

Figure imgf000067_0001

HETCOR-NMR Spectrum of Compound 35

 

Figure imgf000068_0001

 

 

3-[3-(benzoylamino)-4-hydroxylphenyl] propanoic acid 35:

 

To a solution of 32 (222 mg, 1.06 mmol, leq.) dissolved in THF (20 mL) was added the catalyst 10 % palladium-on-charcoal (15 % by mass, 33 mg). The resulting mixture was then placed on a hydrogenator, flushed (5 times) with hydrogen and left to agitate under pressure (36 psi.) overnight (12 hrs) while recharging hydrogen pressure twice (36 psi.) until hydrogen up-take by reaction mixture stopped (pressure did not decrease for 1-2 hrs.). The reaction mixture was vacuum filtered through Celite ‘ rinsing with THF. To the filtered solution containing 33 was directly added BzCl (154 mg, 1.1 mmol, 1 eq.) and left to stir at room temperature for 30 min. Then 10 % HCl (25 mL) was added and stirring continued an additional 5 min. followed by extraction with CIT2Cl2 (2 x 35 mL). The organic fractions were combined, dried (MgSO4), and evaporated off solvent. The resulting mixture was re-crystallized with Hexane/ Acetone to afford an off white solid (250 mg) with an 83 % yield from compound 32. Molecular Formula – C16Hi5NO4. Formula Weight – 285.295 g mole“1.

FT-IR (KBR disk) cm” 1 : 3201 (NH, OH), 1692 (CO2H), 1636 (NHAc).

1H-NMR (Acetone-D6) δ: 2.60 (t, 2H, J = 7.4, H- 3), 2.84 (t, 2H, J = 7.9, H-2), 6.89 (d, IH, J = 8.2, H-8), 7.00 (dd, IH, J = 2.1 , 8.25, H- 9), 7.57 (m, 4H, H-5, H-4′, H-5′, H-61), 8.05 (d, 2H, J = 8.2, H-3′, H-7′), 9.07 (broad s, IH, NH), 9.54 (broad s, IH, OH), 10.58 (broad s, IH, CO2H).

13C-NMR (Acetone- D6) δ: 30.87 (C- 3), 36.21 (C- 2), 118.69 (C- 8), 123.31 (C-5), 123.41 (C- 6), 126.88 (C- 9), 127.37 (C- 4), 128.54 (C-41, C-61), 129.61 (C-31, C-7′), 132.99 (C-51), 134.99 (C-21), 148.03 (C-7), 167.34 (C-I1), 173.94 (C-I ).

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1H-NMR Spectrum of Compound (+/-V36

Figure imgf000069_0001
Figure imgf000069_0002

13 C-NMR Spectrum of Compound (+/-V36

Figure imgf000070_0001
Figure imgf000070_0002

N-(l-oxaspiro[4.5]deca-6,9-dien-2,8-dion-7-yl)acetamide (+/-)-36: To a solution of 34 (122 mg, .547 mmol, 1 eq.) dissolved in acetone (10 mL, 0 0C) was added PIFA (306 mg, .71 1 mmol, 1.3 eq.) in one portion and stirred for 20-25 minutes (confirmed by tic: [1 : 1] EtOAc/Hexane). The reaction mixture was diluted with ethyl acetate (15 mL), washed with cold water (10 mL), dried organic fraction (MgSO4) and evaporated off solvent to afford a Tan solid. The crude product was purified by re-dissolving with CHCI3, filtering of the solution through Celite ®, evaporating off the solvent and placing it under vacuum overnight to afford an off white solid (120 mg, 98 % yield). Molecular Formula – C1 1Hi iNO4. Formula Weight – 221.209 g mole“1. FT-IR (KBR disk) cm“1: 3333 (NH), 1777 (lactone), 1668 (amide), 1650 (ketone), 1620 (α, β-conjugation to ketone). 1H-NMR (CDCl3) δ: 2.17 (s, 3H, H-2′), 2.44 (m, 2H, H-4), 2.81 (m, 2H, H-3), 6.35 (d, IH, J = 10.0, H-9), 6.94 (dd, IH, J = 3.1, 10.0, H- 10), 7.75 (d, IFI, J = 3.1, H-6), 7.99 (broad s, IH, NH). 13C-NMR (CDCl3) δ: 24.86 (C- 2′), 28.36 (C- 4), 32.91 (C- 3), 79.76 (C-5), 124.30 (C- 6), 127.12 (C- 9), 131.55 (C- 7), 148.37 (C-10), 169.51 (C-I’), 175.46 (C-2), 179.40 (C- 8).

 

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1H-NMR Spectrum of Compound 32

Figure imgf000059_0001
Figure imgf000059_0002

(2E)-3-(4-hydroxyl-3-nitrophenyl) acrylic acid 32: To a solution of 4- hydroxyl-3-nitrobenzaldehyde (1.073 g, 6.43 mmol, 1 eq.) dissolved in pyridine (25 mL) was added piperidine (25 drops) and the resulting mixture was stirred (4-5 min.). Malonic acid (1.671 g, 16.1 mmol, 2.5 eq.) was then added in one portion and the resulting mixture was warmed (60-63 0C) and stirred overnight (12-14 hrs, confirmed by tic: EtOAc, mini work up, 10 % HCl and EtOAc). The reaction was cooled and acidified (50 % HCl) until yellow precipitate formed (pH~2). This yellow precipitate was extracted with ethyl acetate (2 x 150 niL). The organic fractions were combined and washed with brine (150 mL), dried (MgSO4), and the solvent was evaporated to afford a yellow solid. Removed excess solvent by vacuum and used without further purification (1.250 g, 93 % yield). Molecular Formula – CgH7NO5. Formula Weight – 209.156 g mole“1. FT-IR (KBR disk) cm“1: 2942 (OH), 1684 (CO2H), 1626 (C=C), 1533,1270 (NO2). 1FI-NMR (Acetone-D6) δ: 2.87 (broad s, IH, OH), 6.58 (d, IH, J= 16.0, H-2), 7.27 (d, IH, J= 8.8, H-8), 7.70 (d, IH, J= 16.4, H-3), 8.08 (d, IH, J= 2.2, 8.5, H-9), 8.40 (d, IFI, J = 2.2, FI-5), 10.67 (broad s, I H, CO2FI). The13C-NMR of this compound agrees with the previously published data.52

 

 

 

Con Dao Island, Vietnam

 

con dau six senses resort image

Con Dao Island, Vietnam

 

This 16-island archipelago is a “pocket of paradise,” says Robert Reid, a travel editor at Lonely Planet.

Getting there: Take a 45-minute flight from Ho Chi Minh City.

What to do: The diving is among the best in Vietnam. Take scuba lessons as a couple or discover the nearby secluded beaches of Bai Dat Doc and Dam Trau.

Where to stay: Six Senses resort offers luxury villas on the East Vietnam Sea. The resort has an in-house spa offering traditional Vietnamese healing practices; it also boasts outdoor treatment rooms and a yoga and meditation pavilion. Inquire for rates.

 

Con Dao travel guide – Wikitravel

wikitravel.org/en/Con_Dao

Con Dao is an island off the southern coast of Vietnam. … The Con Dao Islands separated from the mainland about 15,000 years ago. This has resulted in the …

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NMR Structure Elucidation of Small Organic Molecules and Natural Products: Choosing ADEQUATE vs HMBC

 Uncategorized  Comments Off on NMR Structure Elucidation of Small Organic Molecules and Natural Products: Choosing ADEQUATE vs HMBC
Jun 092015
 
Abstract Image

Long-range heteronuclear shift correlation methods have served as the cornerstone of modern structure elucidation protocols for several decades. The 1H–13C HMBC experiment provides a versatile and relatively sensitive means of establishing predominantly 3JCHconnectivity with the occasional 2JCH or 4JCH correlation being observed. The two-bond and four-bond outliers must be identified specifically to avoid spectral and/or structural misassignment. Despite the versatility and extensive applications of the HMBC experiment, it can still fail to elucidate structures of molecules that are highly proton-deficient, e.g., those that fall under the so-called “Crews rule”. In such cases, recourse to the ADEQUATE experiments should be considered. Thus, a study was undertaken to facilitate better investigator understanding of situations where it might be beneficial to apply 1,1- or 1,n-ADEQUATE to proton-rich or proton-deficient molecules. Equipped with a better understanding of when a given experiment might be more likely to provide the necessary correlation data, investigators can make better decisions on when it might be advisible to employ one experiment over the other. Strychnine (1) and cervinomycin A2 (2) were employed as model compounds to represent proton-rich and proton-deficient classes of molecules, respectively. DFT methods were employed to calculate the relevant nJCHheteronuclear proton–carbon and nJCC homonuclear carbon–carbon coupling constants for this study.

NMR Structure Elucidation of Small Organic Molecules and Natural Products: Choosing ADEQUATE vs HMBC

† Discovery and Preclinical Sciences, Process and Analytical Chemistry, NMR Structure Elucidation, Merck Research Laboratories, Kenilworth, New Jersey 07033, United States
‡ Discovery and Preclinical Sciences, Process and Analytical Chemistry, NMR Structure Elucidation, Merck Research Laboratories, Rahway, New Jersey 07065, United States
J. Nat. Prod., 2014, 77 (8), pp 1942–1947
DOI: 10.1021/np500445s
*Tel: 908-740-3990. Fax: 908-740-4042. E-mail: alexei.buevich@merck.com.
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Using HMBC and ADEQUATE NMR Data To Define and Differentiate Long-Range Coupling Pathways: Is the Crews Rule Obsolete?
It is well known that as molecules become progressively more proton-deficient, structure elucidation becomes correspondingly more challenging. When the ratio of 1H to 13C and the sum of other heavy atoms falls below 2, an axiom that has been dubbed the “Crews rule” comes into play. The general premise of the Crews rule is that highly proton-deficient molecules may have structures that are difficult, and in some cases impossible, to elucidate using conventional suites of NMR experiments that include proton and carbon reference spectra, COSY, multiplicity-edited HSQC, and HMBC (both 1H–13C and 1H–15N). However, with access to modern cryogenic probes and microcyroprobes, experiments that have been less commonly utilized in the past and new experiments such as inverted 1JCC 1,n-ADEQUATE are feasible with modest sized samples. In this light, it may well be time to consider revising the Crews rule. The complex, highly proton-deficient alkaloid staurosporine (1) is used as a model proton-deficient compound for this investigation to highlight the combination of inverted 1JCC 1,n-ADEQUATE with 1.7 mm cryoprobe technology.

Using HMBC and ADEQUATE NMR Data To Define and Differentiate Long-Range Coupling Pathways: Is the Crews Rule Obsolete?

Gary E Martin
† Discovery and Preclinical Sciences, Process and Analytical Chemistry, Structural Elucidation Group, Merck Research Laboratories, Kenilworth, New Jersey 07033, United States
‡ Discovery and Preclinical Sciences, Process and Analytical Chemistry, Structural Elucidation Group, Merck Research Laboratories, Rahway, New Jersey 07065, United States
§ Discovery and Preclinical Sciences, Process and Analytical Chemistry, Structural Elucidation Group, Merck Research Laboratories, Summit, New Jersey 07901, United States
J. Nat. Prod., 2013, 76 (11), pp 2088–2093
DOI: 10.1021/np400562u
Publication Date (Web): November 6, 2013
Copyright © 2013 The American Chemical Society and American Society of Pharmacognosy
*Phone: 908-473-5398. Fax: 908-473-6559. E-mail: gary.martin2@merck.com.
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KETO ENOL TAUTOMERISM AND NMR

 spectroscopy, Uncategorized  Comments Off on KETO ENOL TAUTOMERISM AND NMR
Jun 032015
 

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A Partial NMR Spectrum of 2,4-Pentanedione

 

 

 

 

 

 

 

Patent EP0922715B1 – Stimuli-responsive polymer utilizing keto …

Carbonyl compounds (aldehydes, ketones, carboxylic esters, carboxylic amides) react aselectrophiles at the sp2 hybridized carbon atoms and as nucleophiles if they contain an H-atom in the α-position relative to their C=O or C=N bonds. This is because this H is acidic and it can be removed by a base leaving behind an electron pair for nucleophilic attacks.

For most compounds in organic chemistry all the molecules have the same structure – even if this structure cannot satisfactory represented by a Lewis formula – but for many compounds there is a mixture of two or more structurally distinct compounds that are in rapid equilibrium. This phenomenon is called tautomerism.

Tautomerism is the phenomenon that occurs in any reaction that simply involves the intramolecular transfer of a proton. An equilibrium is established between the two tautomers (structurally distinct compounds) and there is a rapid shift back and forth between the distinct compounds.

A very common form of tautomerism is that between a carbonyl compound containing an αhydrogen and its enol form (Fig. I.1).

Fig. I.1: A keto-enol reaction
Fig. I.1: A keto-enol reaction

 

An enol is exactly what the name implies: an ene-ol. It has a C=C double bond (diene) and an OH group (alcohol) joined directly to it.

Notice that in the above reaction as in any keto-enol reaction there is no change in pH since a proton is lost from carbon and gained on oxygen. The reaction is known as enolization as it is the conversion of a carbonyl compound into its enol.

Notice also that in the above reaction the product is almost the same as the starting material since the only change is the transfer of one proton and the shift of the double bond.

In simple cases (R2 = H, alkyl, OR, etc.) the equilibrium of the keto-enol reaction lies well to the left (keto structure) (Table I.1). The reason can be seen by examining the bond energies in Table I.2.

 

Compound

Enol Content, %

Acetone

6 * 10-7

PhCOCH3

1.1 * 10-6

CH3CHO

6 * 10-5

Cyclohexanone

4 * 10-5

Ph2CHCHO

9.1

PhCOCH2COCH3

89.2

Table I.1: The enol content of some carbonyl compounds

 

If keto-enol reactions are common for aldehydes and ketones why don’t simple aldehydes and ketones exist as enols?

IR and NMR Spectra of carbonyl compounds show no signs of enols. The equilibrium lies well over towards the keto form (the equilibrium constant k for cyclohexanone is about 10-5).

 

Bond (Energy, kJ/mol)

Sum ( kJ/mol)

keto form

C-H (413)

C-C (350)

C=O (740)

1503

enol form

C=C (620)

C-O (367)

O-H (462)

1449

Table I.2: Bond energies in the keto and in the enol form. The keto form is thermodynamically more stable than the enol form by approximately 50 kJ/mol

The approximate sum of the bond energies in the keto form is 1503 kJ/mol while in the enol form 1449. Therefore, the keto form is thermodynamically more stable than the enol form by approximately 50 kJ/mol.

In most cases, enol forms cannot be isolated since they are less stable and are formed in minute quantities. However, there are some exceptions and in certain cases a larger amount of the enol form is present and it can be even the predominant species:

  • Molecules in which the enolic double bond is in conjugation with another double bond (cases are shown in Table I.1 like Ph2CHCHO and PhCOCH2COCH3)
  • Molecules that contain two or more bulky aryl groups (Fig. I.2). Compound I in Fig. I.2 (a substituted enol) is the major species in equilibrium (~95%) while the keto form is the minor species (~5%). In cases like this steric hindrance destabilizes the keto form (the two substituted aryl groups are 109° apart) while in the enol form 120° apart.

 

Fig. I.2: A keto-enol reaction. The enol form (I) is the major species since the keto form is destabilized by steric hindrance (the substituted aryl groups are closer in the keto form – the C-C angle is 109° and this is unfavorable due to steric hindrance)
Fig. I.2: A keto-enol reaction. The enol form (I) is the major species in this case since the keto form is destabilized by steric hindrance (the substituted aryl groups are closer in the keto form – the C-C angle is 109° and this is unfavorable due to steric hindrance)

 

Is there experimental evidence that keto-enol reactions are common for aldehydes and ketones?

If the NMR spectrum of a simple carbonyl compound in D2O is obtained – such as pinacolone’s (CH3)3CCOCH3 – the signal for protons next to the carbonyl group very slowly disappears. The isolated compound’s mass spectrum (after the above mentioned reaction with D2O is over) shows that those hydrogen atoms have been replaced by deuterium atoms. There is a peak at (M+1)+ or (M+2)+ or (M+3)+ instead of M+. The reaction is shown in Fig. I.3:

 Fig. I.3: Evidence for a keto-enol reaction when pinacolone (CH3)3CCOCH3 reacts with D2O. When the enol form of the pinacolone reverts to the keto form it picks up a deuteron instead of a proton because the solution consists almost entirely of D2O.
Fig. I.3: Evidence for a keto-enol reaction when pinacolone (CH3)3CCOCH3 reacts with D2O. When the enol form of the pinacolone reverts to the keto form it picks up a deuteron instead of a proton because the solution consists almost entirely of D2O.

 

What mechanism can be proposed for the above reaction?

Enolization is a slow process in neutral solution, even in D2O, and is catalyzed by acid or base in order to happen.

In the acid-catalyzed reaction the molecule is first protonated on oxygen and then loses the C-H proton in a second step (Fig. I.4). When the enol form reverts to the keto – since this is an equilibrium process – it picks up a deuteron instead of a proton since the solution is D2O.

 

Fig. I.4: The acid-catalyzed keto-enol reaction mechanism. If D2O is the solvent then the α-hydrogens to carbonyl group are replaced by deuterium.
Fig. I.4: The acid-catalyzed keto-enol reaction mechanism. If D2O is the solvent then the α-hydrogens to carbonyl group are replaced by deuterium.

In the base-catalyzed reaction the C-H proton is removed first by the base (for example hydroxide ion OH, OD in our case) and the proton (or D+ in our case) added to the oxygen atom in a second step (Fig. I.5).

Fig. I.5: The base-catalyzed keto-enol reaction mechanism. If D2O is the solvent then the α-hydrogens to carbonyl group are replaced by deuterium.
Fig. I.5: The base-catalyzed keto-enol reaction mechanism. If D2O is the solvent then the α-hydrogens to carbonyl group are replaced by deuterium.

Notice that the enolization reactions in Fig. I.4 and Fig. I.5 are catalytic. In the acid-catalyzed mechanism the D+ (or H+ if water is the solvent) is regenerated at the end (catalyst). In the base-catalyzed mechanism OD (or OH if water is the solvent) is regenerated at the end (catalyst).

The enolate ion generated from the enol (Fig. I.6) in the base-catalyzed mechanism is nucleophilic due to:

  • Oxygen’s small atomic radius
  • Formal negative charge

An enolate ion is an ion with a negative charge on oxygen with adjacent C-C double bond.

 

 Fig. I.6: Enolate ion resonance contributors. Although the major contributor is resonace structure I when it reacts as a nucleophile structure II is more reactive.
Fig. I.6: Enolate ion resonance contributors. Although the major contributor is resonace structure I when it reacts as a nucleophile structure II is more reactive.

Enolates are reactive nucleophiles. Although the major enolate Lewis contributor shows concentration of electron density on the electronegative oxygen when it reacts as a nucleophile, it behaves like the electron density is concentrated on the α-carbon next to carbonyl group.

Enolates react with alkyl halides, aldehydes/ketones and esters and these reactions are shown in the post entitled “The chemistry of enolate ions – Enolate ion reactions”.


 

References
  1. A.J. Kresge, Pure Appl. Chem., 63, 213 (1991)
  2. B. Capon, The Chemistry of Enols, Wiley, NY, 307–322 (1990)
  3. S.E. Biali et al., J. Am. Chem. Soc. 107, 1007 (1985).

 

 

 

 

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http://www.slideshare.net/chemsant/nmr-dynamic

 

 

http://article.sapub.org/10.5923.j.ajoc.20140401.01.html

2-fluoro-3-hydroxycyclopent-2-enone and 2-fluoro- 1,3-cyclopentanedione (1c): This compound was obtained as a 52:48 mixture of keto-enol and diketo tautomers in 50% yield as a yellow-brown solid, mp 70-72°C. NMR:1H: δ 2.36 (t, 3JH-H = 16.2 Hz, 2H), 2.85 (m, 2H), 5.91 (d, 2JH-F = 47.7 Hz, 1H). 13C: δ31.1, 90.8 (d, 1JC-F = 251.3 Hz), 122.3 (d, 1JC-F = 233.9 Hz), 210.1 (d, 2JC-F = 31.0 Hz). 19F: keto-enol: δ-161.4 (s, 1F); diketo: δ-195.5 (d, 2JF-H = 47.7 Hz, 1F). Analysis calcd for C5H5FO2: C, 51.73, H, 4.34. Found: C, 51.48, H, 4.31.

 

 

 

 

 

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NMR Structural Techniques’ Contribution To The Drug Discovery And Development Process

 spectroscopy  Comments Off on NMR Structural Techniques’ Contribution To The Drug Discovery And Development Process
Jun 012015
 

Carla Marchioro

Introduction

As is well known the drug discovery and development process is a complex process typically starting from the target identification and validation of a target to progress to clinical studies and hopefully ending with a new drug to the market [Figure 1].

 

 

In this process, different approaches and methods are required to understand the disease’s mechanisms, to profile hit molecules that will be progressed to leads suitable for full scale lead optimization programmes and then to generate quality drug candidates to advance to clinical studies.

Focusing on small molecules’ drugs and on hit to candidate phases, a variety of techniques will be used to study the compounds’ profiles at different levels including the physicochemical profiles, purity, solid state behaviours and structures to ensure a quality hit/lead/candidate and related data to allow understanding of the mechanism of action and SAR correlation.

Nuclear Magnetic Resonance (NMR) spectroscopy will play a pivotal role generating data on the molecular interactions between ligands and biological targets, in addition to providing the structures of drug molecules, by-products, impurities, metabolites and quantification data.

NMR Screening Impact 

During the drug discovery phase, NMR spectroscopy is becoming more and more relevant with application at multiple stages along the progression of a project: NMR experiments are used for hits generation, lead discovery and optimization, evaluation of in vitro/in vivo selectivity and efficacy, studies drug toxicity profiles and identification of new drug discovery targets.

Over the last years there has been a large increase in the application of NMR techniques for the rapid determination of protein-ligand structures and interactions, to powerfully screen fragment-based libraries, to identify biological relevant ligand interactions, and to monitor changes in the metabolome from bio-fluids and cells to explore compounds activity.

Focusing on the NMR-based screening techniques, the NMR experiments could be divided into two main categories: target observed and ligand observed methods.

Without doubt, high resolution protein structure is a key requirement to evaluate the biological relevance of a hit from screening and HTS (high-throughput screening) and NMR together with X-ray are playing an essential role.

The last period has witnessed the generation of fast NMR sequences and methods to allow a faster impact on the drug discovery project time but the NMR target-observed techniques still require time, material, possible labelling and difficulties in handling a number of different hits and studies on mixtures.

Nevertheless, if the resonance assignment of the labelled target is known, the exploitation of differences in chemical shifts between free and bound target in two dimensional correlation spectra (shift mapping) will provide important structural information on the site of binding. The experiments could be also be of high value on selectively labelled target decreasing the spectra complexity and so increasing the size of the target that could be studied by NMR techniques.

Considering now that the chemical shift is highly sensitive to the environment of the atom and, as a consequence, it provides information on the binding of a small molecule to a biological target, and on which part of the molecule is interacting and where, it is clear that ligand-observed techniques could generate proof and data for the binding understanding and profile.

In addition, other experiments based on molecule relaxation values are sensitive to the motion of the compound (free vs bound state) and together these experiments will allow validation of ligand binding and/or identification of ligands also in mixtures.

The ligand observed techniques benefit of:

  • one-dimensional experiments;
  • detection of the ligand’s signals (facilitating also the mixture analysis);
  • smaller amount of target substrate;
  • structural and binding information of the ligand;
  • detection of week binding ligands;
  • limited restrictions on size and type of the target, with no isotope labelling requirement or target information details.

On the undesirable side, the techniques could generate false negatives (strong binding and slow exchange equilibria) or false positives (unspecific binding) but all these aspects could be further studied to result in a substantiated answer.

During the Hits generation phase, NMR will be used for the determination of binding affinity values toward the hit validation step to generate a lead where the NMR experiments will also remove the false positives and locate the binding site (for example within the FBDD approach). In the lead optimization phase, to improve potency of the compounds, the epitope mapping will be determined, together with the conformation of the bound ligand, while in the late lead optimization stage for the candidate selection the NMR will support the bioavailability, ADME, PK and toxicological experiments.

The combination of NMR screening methods with other techniques, such as in silico computational protocol, X-ray crystallography, and biophysical experiments will decrease the number of compounds to be studied generating filters and resulting in time and cost saving and efficiency increase. The NMR will be so used to screen and profile a library (or set) of compounds with the unique ability of providing proof for binding between the ligand and the biological target and subsequently being able to detect the binding site and determining the construct of the complex.

This short note will not include technical details of the many NMR-based methods that could be found in several papers and reviews across the last decade [1-8].

The versatility of the NMR techniques is allowing the detection of target-ligand interactions through a large variety of measurements. The insights will derive from the observation of peak intensity and/or line-width changes, saturation transfer differences (STD), chemical shifts perturbations, R2 relaxation effects, R1, sel competition data, induced transferred NOEs, interligand NOEs, diffusion coefficient measurements and changes, and, in general, from monitoring any changes in the NMR spectra resulting from the ligand-target interactions.

A big impact of the NMR techniques is also evident on the “undruggable” targets when other techniques alone fail to result in relevant data and studies on protein-protein, protein-membrane macromolecular recognition are now becoming more and more frequently successfully progressed [9].

The lead compound will need then to be optimized in the bioavailability, efficacy and toxicity profile to result in a candidate to be progressed to in vivo studies, in animals, and finally on humans.

NMR will contribute heavily in all these phases will full characterization of the compound and solid state data, stability studies, formulation studies and NMR-based metabolomics experiments. All these aspects will be covered in a future contribution.

References 

1. M. Pellecchia, I. Bertini, D. Cowburn, C. Dalvit, E. Giralt, W. Jahnke, T.L. James, S.W. Homans, H. Kessler and C. Luchinat, Nat. Rev. Drug Discovery, 7, 738 (2008).

2. R. Powers, Expert Opin. Drug Discov., 4(10), 1077 (2009).

3. R. Powers, J. Med. Chem., 57(14), 5860 (2014).

4. M.J. Harner, A.O. Frank and S.W.Fesik, J. Biomol. NMR, 56(2), 65 (2013).

5. C. Dalvit, Prog. Nucl. Magn. Reson. Spectrosc., 51, 243 (2007).

6. M. Mayer and B. Meyer, Angewandte Chemie Int. Edition, 38,1784 (1999).

7. P.J. Hajduk, D.J. Burns, Comb. Chem. High Throughput Screen., 5, 613 (2002).

8. W.Jahnke and D.A. Erlanson (Editors), Fragment-based Approaches in Drug Discovery, Wiley-VCH, 2006.

9. D.M. Dias, I. Van Molle, M.G.J. Baud, C. Galdeano, C.F. G. C. Geraldes and Alessio Ciulli, ACS Med. Chem. Lett., 5 (1), 23 (2014).

In Part 2 of this series, Carla Marchioro continues to offer her insights into the contribution of NMR structural techniques to the drug discovery and development process.

 

Introduction 

After some insights on the impact of NMR techniques on the initial drug discovery phase [1], NMR techniques applied in the progression of a compound from lead to candidate and to drug are clearly having, together with other techniques, a large impact with full structural determination, full understanding of chemical reactions, studies of molecules’ behaviour in solutions and solid states and stability monitoring with determination of by-products.

NMR Techniques in Lead Optimization and Drug Development 

As soon as a compound has been identified as a lead to be progressed to the candidate phase, several NMR studies will be required to support the chemical effort, and to ensure a quality profile of the selected compound.

Synthesis of different compounds will be progressed to obtain the desired biological profile and structures will be characterized and studied to also support the computational effort, and to monitor and determine the purity for the biological tests.

Several techniques will be used, such a MS, IR, HPLC,…, to results together with the NMR data in a full profile of the studied compound.

Classical mono- and two-dimensional NMR techniques (1H and 13C) will be performed and, if required, experiments on additional nuclei will add further information to the full structural determination. As an example, in Figures 1 and 2, 1H-15N g-HNMQC, 19F-15N g-HNMQC, and 1H-29Si g-HMQC have been used to obtain the full structures characterizations [2, 3].

 

Figure 1: 1H-15N g-HNMQC and 19F-15N g-HNMQC experiments.

Figure 1: 1H-15N g-HNMQC and 19F-15N g-HNMQC experiments.

 

 

 

Figure 2: 1H-29Si g-HMQC experiment.

Figure 2: 1H-29Si g-HMQC experiment.

 

The selected compound(s) will be moved to candidate development with scale-up of the synthetic route, and characterization of the resulting material.

NMR will play an important role in reaction monitoring to ensure, with other techniques, a full understanding of the different steps of the chemical steps with identification of by-products and impurities.

Hyphenated HPLC- NMR has been used in the example in Figure 3 for the identification of co‑eluting low‑level impurities in key intermediate; Spectrum A has been acquired after injection of the mother liquors while Spectrum B has been acquired after injection of 100 µL of a solution of key-intermediate. Detailed analysis on the impurity in the mother liquors with a time-slice HPLC-NMR experiment (3 spectra at 10 sec. interval during peak elution) allowed the confirmation that the impurity was in fact a mixture of two co-eluting products. Structures determination has then been obtained after purification using standard NMR experiments [2].

 

Figure 3: Identification of co-eluting low-level impurities.

Figure 3: Identification of co-eluting low-level impurities.

 

Critical experiments are also required in the case of UV transparent compounds, which will not be monitored by classical chromatographic techniques as reported in Figure 4 [2].

 

Figure 4: Reaction monitoring: Continuous-flow HPLC-NMR.

Figure 4: Reaction monitoring: Continuous-flow HPLC-NMR.

 

The final API will be fully characterized to profile the solid state profile, and to support the formulation studies. In addition to the solution phase NMR, solid-state NMR (ssNMR) will be used together with a variety of techniques to ensure a full understanding of compound behaviour.

An interesting application of solution NMR is reported in Figure 5 where experiments have been progressed for the determination of the critical micelle concentration (CMC) (value of the solute concentration at which half the total solute is present in the free monomeric form). NMR spectroscopy can be an alternative method to measure the CMC value, being the chemical shift concentration-dependent, particularly in the case of solute-solute intermolecular interactions, with typical downfield shifts of 1H NMR resonances on dilution.

 

Figure 5: Critical Micelle Concentration (CMC) Determination.

Figure 5: Critical Micelle Concentration (CMC) Determination.

In the example, the particularly large shielding for the aromatic protons allowed the assumption that the aromatic rings of the studied molecules that constitute the aggregate are placed in the inner hydrophobic part of the micelle, while the N-acetylpiperazine ring is somehow representing the hydrophilic external surface of the micelle itself. The forces that are involved in the aggregation are then those typical of π-staking. The CMC can then be evaluated plotting the chemical shift variation (Δδ, ppm) versus the reciprocal of the concentration (L/mol). No significant chemical shift variation was observed in the solutions at concentration ≤ 1 mg/mL, while a linear trend was observed in the concentration range 50 ÷ 3 mg/mL. Thus, assumption could be made that the intercepts of these lines on the x axis corresponded to the 1/CMC value. NMR measurements performed at 15 °C, 25 °C, 35 °C and 45 °C allowed the temperature dependence of the CMC to be determined and the thermodynamic parameters of the micellization process to be extrapolated [4].

In Parts 1 and 2, a few examples of the possibilities of the NMR techniques to support the drug discovery and development have been made with a focus on structures determination and characterization. The impact of NMR techniques on in vitro ex vivo in vivo and clinical phases will be covered in Part 3.

References 

1. C. Marchioro, Spectroscopy Solutions , 3 (1), (2015).

2. S. Provera, C. Marchioro, unpublished data.

3. S. Provera, S. Davalli; G. H. Raza; S. Contini; C. Marchioro, Magn. Reson. Chem. , 39, 38 (2001).

4. S. Provera, S. Beato, Z. Cimarosti, L. Turco, A. Casazza, G. Caivano, C. Marchioro, J. Pharm. Biomed. Anal. , 54, 48 (2011).

Carla Marchioro

Scientific Director at R4R & Head of Discovery and Development; Chief Technology & Operations Advisor at AnCoreX

Current
  1. AnCoreX Therapeutics,
  2. R4R
Previous
  1. Aptuit,
  2. GlaxoSmithKline
Education
  1. Università degli Studi di Padova

https://www.linkedin.com/in/carlamarchioro

Carla Marchioro – ResearchGate

www.researchgate.net/profile/Carla_Marchioro

Carla Marchioro is Scientific Director, and Head of the Pharma & Analytical Division at Research for Rent, R4R, Italy where she is now after covering related positions in Aptuit and GlaxoSmithKline R&D where she has been leading multidisciplinary and cross national groups. In addition, she is also Chief Technology & Operations Adviser at AnCoreX Therapeutics.

She is an NMR expert with a chemistry background and has large experience in structural techniques. Over the years, she has developed an extended experience in a large part of the Research & Development process from target identification and progression to NDA filling.

In her group, in addition to classical structural and analytical approaches, state of the art techniques and technologies such as “omics”, computer-assisted drug design, fragments base screening, analytical and preparative SFC, quantitation by NMR, ssNMR methods for cells & tissues and more have been introduced and developed.

In addition to the R4R role, she is a member of a number of Scientific Boards, European and National Research funding bodies; and she has been part of the Scientific Advisory Board of the ProtEra company up to February 2010.She is author of a number of publications and presentations and she is a well-recognized member in the scientific community. She has been a member of the ENC Scientific Board, the chair of the 51” ENC (2010), member of the SMASH Conference Board, and the chair of the SMASH 2013 Conference.

Structural & Analytical expertise in the Drug Discovery, Chemical Development and Pharmaceutical Development Departments (up to transfer to Manufacturing groups). In addition, experience in the drug design and understanding of mechanism of action, metabolic pathways and safety related aspects.

Specialties: full understanding of mechanism of actions; full understanding of chemical and biological pathways; software and hardware design and needs; international experiences crossing countries and cultures.

Experience

Chief Technology & Operations Advisor

AnCoreX Therapeutics

 – Present (1 year 2 months)

Scientific Director & Head of Discovery and Development

R4R

 – Present (2 years 6 months)

Scientific Liaison Director

Aptuit

 –  (1 year 4 months)Verona Area, Italy

Director, Head of Structural & Analytical Scientific Strategy

Aptuit

 –  (2 years 3 months)

Director Analytical Chemistry

Aptuit

 –  (4 months)

Director & Site Head of Verona Analytical Chemistry

GlaxoSmithKline

 –  (1 year 6 months)

Objective of the Verona group was to provide Structural & Analytical expertises to the Verona/Harlow Centre for Excellence in Drug Discovery (Neurosciences CEDD), Chemical Development and Pharmaceutical Development Departments (up to transfer to Manufacturing groups) at the Verona GSK site. In addition, to contribute to the international initiatives of Molecular Drug Discovery (MDR) and Analytical Chemistry.

Director & Site Head of Verona & Zagreb Analytical Chemistry

GSK

 –  (2 years)

Objective of the Verona group was to provide Structural & Analytical expertises to the Verona/Harlow/Zagreb Centre for Excellence in Drug Discovery (Neurosciences CEDD), Chemical Development and Pharmaceutical Development Departments (up to transfer to Manufacturing groups) at the Verona GSK site. In addition, to contribute to the international initiatives of Molecular Drug Discovery (MDR) and Analytical Chemistry.
Objective of the Zagreb group was to provide Structural & Analytical expertises to the Zagreb Centre for Excellence in Drug Discovery (MacrolidesCEDD), and Pharmaceutical Development Departments at the Zagreb GSK site. In addition, to contribute to the international initiatives of Molecular Drug Discovery (MDR) and Analytical Chemistry.

Director

GlaxoWellcome

 –  (1 year)

Honors & Awards

Additional Honors & Awards

Contract Professor, Ferrara University (1999–2003);
Chiar for the SMASH2003 Conference, Verona, Italy
Chair for the 51th Experimental NMR Conference (ENC) (2010, Daytona Beach, US)
Chair for the SMASH2013 Conference, Santiago de Compostela, Spain

Publications

Discovery Process and Pharmacological Characterization of a Novel Dual Orexin 1 and Orexin 2 Receptor Antagonist Useful for Treatment of Sleep Disorders(Link)

Bioorganic & Medicinal Chemistry Letters (2011), 21, 5562

September 1, 2011

A novel, drug-like bis-amido piperidine derivative was identified as a potent dual OX1 and OX2 receptor antagonists, highly effective in a pre-clinical model of sleep.

VERONA,  ITALY

  1. Verona – Wikipedia, the free encyclopedia

    en.wikipedia.org/wiki/Verona

Verona (Italian pronunciation: [veˈroːna] ( listen); Venetian: Verona, Veròna) is a city straddling the Adige river in Veneto, northern Italy, with approximately  …

Map of verona italy.

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