ACD/NMR Workbook: A Toolkit for Pharma’sNMR Spectroscopist

Janet Caceres-CortesBristol-Myers Squibb Company

NMR in the Pharmaceutical Industry

NMR spectroscopy has evolved into an important technique in support of

critical activities within the discovery and development workspace:

SAR and lead optimization, process chemistry,

metabolite ID and quantification, mechanistic studies,

biophysical characterization, stability/degradation,

process optimization, safety assessments …

different structural modalities:

small molecules, millamolecules, proteins

synthetic intermediates and final products, drug

metabolites from in vivo and in vitro sources,

endogenous metabolites in biofluids, excipients,

residual solvent and impurities …

We support

We work with

We analyze

Discovery NMR Experts

DevelopmentNMR Experts

Medicinal Chemists

ProcessChemists

ACD/Labs: Spectrus Processor; 1H, 13C, 15N Predictors;

NMR Workbook, Spectrus DB

ACD/Labs NMR Landscape

ACD/Labs

Desktop Processing/Prediction

Batch Characterization Reports

Metabolite ID Reports

From OA to Email & Electronic Notebook:

PDF w/ Embedded JDX

Journals & Patents

Database 1H, 13C, 15N

1H NMR (DMSO-d6, 600MHz):d = 8.70 (d, J=4.4 Hz, 1H), 7.95(d, J=9.1 Hz, 1H), 7.53 (d, J=4.3Hz, 1H), 7.48 (br. s., 1H), 7.40(dd, J=9.0, 2.1 Hz, 1H)

Samples 1H 13C 15N

1 √ √

2 √ √ √

3 √ √

4 √ √ √

5 √ √

Integrated Work Practices

ACD/NMR Workbook…

Initially adopted for incorporation into metabolite ID workflow and reporting activities

DRUG

M1

M7

M8M10

M9

M3

M4

M5

M6

M2

Metabolite Identification

Drug Discovery: enables early identification of potential metabolic liabilities to help avoid failure at later stages : Soft spot analysis for SAR and lead optimization Assess contributions to pharmacokinetic parameters Toxicity evaluations in preclinical species

Drug Development: a strong emphasis is placed on truly understanding the efficacy and safety profile of drugsand the contributions of their metabolites to these profiles. Metabolite profiling Determination of metabolite exposures Assessment of metabolic clearance pathways in humans and toxicological species MIST - metabolite characterization plan.

8 7 6 5 4 3 2 1 0Chemical Shift (ppm)

9 8 7 6 5 4 3 2 1 0Chemical Shift (ppm)

NMR Metabolite ID Activities

Full Assignment of Parent compound from 1D and 2D spectra

Usually provided from a synthetic batch Available in milligram quantities Impurities left from the reaction mixture, solvents and other sources are small compared to parent NMR peaks

Comparison of metabolite spectra with parent (1D, and 2D when needed)

Generally isolated from in vitro and in vivo sources Available in low microgram quantities Impurities are at scale with or larger than metabolite NMR peaks

13C spectra usually not obtained

Metabolite spectra

Why ACD/NMR Workbook…Efficiency and Ease of useParent datasets:

Eliminates the need to print spectra and assign on paper Full assignments quickly obtained; assignments appear across all spectra

1H and 13C chemical shifts and coupling constants table generated Used for reporting, but also used for elucidating metabolite structures

Flexibility and Fit for PurposeMetabolite datasets:

Tile or overlay of parent and metabolite spectra (e.g. 1H/1H) Tile or overlay of metabolite spectra (e.g. HSQC/HMBC) Assignments by iterative inspection of 1D and 2D data accommodated Can utilize dark regions in 2D to tag impurity peaksPartial or targeted assignments for “fit for purpose” work practices (e.g. 1H spectrum yields an unambiguous structure or key correlations are established in a dataset where impurities preclude a full elucidation)

ACD/NMR Workbook

Easy access to data and structures; also can see experiment type

Easy access ‘one click’ buttons for routine tasks

Drag parent data into a project for assigning (can have multiple projects open)

Undo/redo buttons

1d spectra height adjusted with mouse scroll

Assignment numbers shown on vertical and horizontal axes

Create horizontal and vertical lines in 2D data to facilitate peak picking

ACD/NMR WorkbookACD v12

ACD v2012

ACD 12 peak picking sometime results in small chemical shift differences for the same carbon- program treats them as different carbons which can cause issues with table generation

ACD v2012 – can click to add horizontal lines, vertical lines or both

ACD/NMR Workbook

Peak picking can be performed in an automated fashion or manuallyAssignments are easily generated by selecting a peak of interest and scrolling to the corresponding atom on the structure

ACD/NMR Workbook Molecule can be renumbered or changed within the Workbook, without the need to go into ChemSketch.

ACD/NMR Workbook Structure and data windows can be detached from the Workbook providing increased flexibility to the users.

Metabolism and Disposition of BMS-690514

M1, M3, M6, M7, and M9 were the major circulating metabolites in human plasma after oral administration of BMS-690514.

Hong et al . Chem. Res. Toxicol. (2011)24, 125–134

M6 [NMR] M8 [NMR, mixture]

M7 [NMR]

M1 [NMR]

M2

M3

M9

M37

NMRSync allows synchronized peak peaking and assignment across a dataset

Toolbar –quick way to label peaks, define and edit multiplets and assignments Multiplet or peak picking in one spectrum transfers information to the others Chemical shifts are displayed on screen, making assigning easier

1

2

3

4

5

NMRSync generates tables summarizing the assignment information

Assignments, chemical shift, coupling constants and correlations are captured in tables eliminating the need to manually capture this information Eliminates errors associated with typing this information manually Can be copied to report editor or clipboard, exported , and inserted into a Microsoft Word or Excel table

Atom# XHn H Shift H Multiplicity C Shift COSY H HMBC C HMBC

2 CH2 3.069 d (7.23) 58.740 1.95 1.82, 2.92, 3.29, 3.73 73.52, 52.31, 55.30

2 CH2 1.954 br.s 58.740 3.07, 3.29, 2.11 1.82, 2.92, 3.29, 3.73 73.52, 55.30, 52.31

3 CH 3.292 d (3.73) 73.519 1.95, 2.46 1.32, 1.82, 1.95, 2.46, 3.07 58.74

4 CH 2.456 br.s 55.302 1.32, 3.29 1.32, 1.82, 1.95, 2.92, 3.07 32.09, 73.52

5 CH2 1.324 d (9.65) 32.094 1.82, 2.11, 2.46 2.11, 2.46, 2.92 55.30, 52.31, 73.52, 55.88

5 CH2 1.824 d (10.74) 32.094 1.32 2.11, 2.46, 2.92 55.88, 55.30, 58.74, 73.52

6 CH2 2.107 br.s 52.309 1.32, 1.95, 2.92 1.32, 1.95, 3.07, 3.73 32.09

6 CH2 2.923 d (10.74) 52.309 2.11 1.32, 1.95, 3.07, 3.73 58.74, 32.09, 55.30

7 CH2 3.731 br.s 55.883 1.32, 1.82, 6.58 113.58, 115.33, 52.31, 58.74, 114.14

8 C 114.145 3.73, 6.58, 7.47

9 C 115.331 3.73, 7.47

12 CH 7.777 s 148.203 154.47

14 C 154.474 7.78

15 CH 6.576 br.s 113.575 7.47 3.73, 7.47 118.66, 114.14, 55.88

16 CH 7.473 br.s 118.661 6.58 6.58 113.58, 115.33, 114.14

20 C 140.360 7.12, 7.28, 7.31

21 CH 7.282 br.s 109.246 7.12 6.75, 7.12, 7.31 110.88, 160.74, 115.70, 140.36

22 C 160.744 3.79, 6.75, 7.12, 7.28, 7.31

23 CH 6.751 d (7.45) 110.877 7.31 7.12, 7.28, 7.31 115.70, 109.25, 160.74

24 CH 7.308 t (8.79, 8.79) 131.060 6.75, 7.12 7.12 160.74, 109.25, 110.88, 140.36, 115.70

25 CH 7.118 d (7.23) 115.700 7.31, 7.28 6.75, 7.28, 7.31 140.36, 131.06, 110.88, 109.25, 160.74

27 CH3 3.793 s 56.233 160.74

Comparisons of parent and metabolite 1H spectra are usually performed first Tile by selecting the appropriate icon and setting the number of spectra to display. Parent spectra is displayed with assignments facilitating comparisons

Tile or overlay spectra for easy comparisons and to assist metabolite assignments

Overlay spectra by clicking on Series – Collect Spectra The spectra can be offset (shown here) or superimposed using the Manual Offset tool Parent spectra is displayed with assignments facilitating comparisons

Tile or overlay spectra for easy comparisons and to assist metabolite assignments

Tile or overlay spectra for easy comparisons and to assist metabolite assignments

Overlay spectra by clicking on Series – Collect Spectra The spectra can be offset (shown here) or superimposed using the Manual Offset tool Parent spectra is displayed with assignments facilitating comparisons

Comparisons of parent and metabolite COSY and HSQC are performed if the metabolite structure is not determined from its 1H spectrum. In overlay mode, spectra are distinguished by color and different font size Peak picking and assignments can be performed in overlay mode

Tile or overlay spectra of 2D data from Parent and Metabolite

ParentMetabolite

In Tile mode the cursor spans both tiles facilitating comparisons Peak picking and assignments can be performed in tile mode

Parent M1

Tile or overlay spectra of 2D data from Parent and Metabolite

Dark Regions are regions in the spectrum that are not taken into account by the program when performing peak picking, integration, and verification. Solvent and impurity peaks can be tagged in both the 1D and 2D spectra Very useful when assigning around regions with a lot of extraneous peaks

Dark Regions used to tag impurity peaks

After assignments are complete, dark regions can be removed or hidden. To hide a dark region without removing it, uncheck the Active box in the Dark Region Organizer.

Dark Regions can be hidden

Comparing COSY, HSQC and HMBC spectra from the metabolite facilitates iterative peak peaking to complete assignments

Tiling HSQC and HMBC spectra from the metabolite to complete assignments

8Hz 4Hz

Changing Structures Mid-Stream

Can use parent molecule or a proposed structure when beginning metabolite ID work. Structures can be changed in ChemSketch and re-incorporated into the NMR Workbook project

21

25

16

8.5 8.0 7.5 7.0 6.5 6.0Chemical Shift (ppm)

211523 16

1224

16

25

21

Easily incorporate assigned spectra and all associated data into reports

Ideal for comprehensive metabolite ID reports: ACD numbered structure,1D spectra of parent and metabolite with assignments, 2D spectra and structures with key correlations highlighted, Table of chemical shifts

4

3

5

6

N1

2

7

15

8

16

N10

9

N11

12

N13

14

N19

25

24

20

21

22

23

O26

CH3

27

OH17

NH218

OH28

4

3

56

N1

2

7

15

8

16

N10

9

N11

12

N13

14

NH19

25

24

20

2122

23

O26

CH3

27

OH17

NH218

1D ROE

ACD/NMR Workbook NMR Workbook is more efficient than assigning on spectral printouts

Syncing spectra in tile or overlay mode is central to metabolite ID work, where comparisons to parent data is routinely performed.

Dark regions can be used for ‘tagging’ impurities and are easily hidden for reporting.

Proposed structures can be changed once a definitive structure is elucidated

The Workbook has numerous advantages for reporting and publishing.

The v2012 edition of NMR Workbook has significant improvements over version 12 that better address the diverse spectral features of NMR datasets and it has an even more simplified user interface