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Spectral Assignments and Reference DataReceived: 30 July 2007 Revised: 19 September 2007 Accepted: 26 September 2007 Published online in Wiley Interscience:

(www.interscience.com) DOI 10.1002/mrc.2122

Complete 1H and 13C assignments of (21R) and(21S) diastereomers of argatrobanDiego Colombo,a∗ Patrizia Ferraboschi,a Paride Grisentib andLaura Legnanic

The complete 1H and 13C NMR assignments are reported for the antithrombotic (21R)- and (21S)-argatroban by 1D and 2D NMRexperiments (HSQC, HMBC, NOESY and 1H– 1H COSY). Some well-resolved signals could be used for an accurate measurementof the diastereomeric composition of argatroban. Copyright c© 2008 John Wiley & Sons, Ltd.

Keywords: NMR; 1H; 13C; 1D/2D NMR; thrombin; inhibitor; diastereomeric ratio

Introduction

Argatroban is a synthetic inhibitor of thrombin (the serine proteasethat catalyzes fibrin formation and platelet aggregation) that playsa relevant role in the thrombotic vascular disease.[1] The mostfrequently prescribed anticoagulant with antithrombin activityis heparin but limitations due to its chemical heterogeneity andwidespread binding to proteins and endothelial cells, in addition toseveral adverse events as the heparin-induced thrombocytopenia(HIT), prompted to investigate the design of low-molecular-weight,selective inhibitors of thrombin. Argatroban is a small molecule(MW 509) that possesses unique properties, which may make itpreferable to other anticoagulants because of selectivity for thecatalytic site of thrombin, ability to bind and inhibit clot-boundthrombin, short half-life and reversible nature of binding as wellas a predictable dose–response relationship.[2]

Chemically, argatroban is the dipeptide between arginine and4-methyl-2-piperidine carboxylic acid; the NH2 group of arginineis bonded to a methyltetrahydroquinoline sulfonyl group. Fourstereogenic centers are present in the molecule: in the courseof the synthesis[3,4] optically pure L-arginine and (2R, 4R)-4-methyl-2-piperidine carboxylic acid are employed, leading to adipeptide with three stereochemically defined stereocenters. Theadditional stereocenter at C-21 is introduced in the course ofthe final step; reaction of the suitably protected dipeptide withthe sulfonyl chloride of 4-methylquinoline and hydrogenationof heteroaromatic ring, with concomitant removal of protectinggroups, leads to a mixture of 21-epimers in a roughly 64/36 ratio.21R and 21S configurations were assigned by X-ray studies, andan HPLC method for separation of epimers is reported.[5] Bothisomers are endowed with biological activity, the 21S epimerbeing twice potent as the 21R one with regard to thrombininhibition,[5] and the diastereomeric mixture is applied as drug,without separation, in management of acute coronary syndrome,percutaneous coronary intervention and in treatment of HIT.[2]

The lack of NMR studies (to the best of our knowledge onlythe resonances of aromatic protons have been assigned[6])and the presence of chirality, which plays a critical role inpharmaceutical research and development,[7] prompted us toassign the 1H and 13C NMR resonances of (21R) (1a) and (21S)(1b) diastereomers of the broadly used substitute for heparin,

argatroban. The experimental results were also supported bycalculations.

N NH

NH2

H3C

HOOC O

HNSO2

NH

HN

R'

R

1

15 20

2

1617

19

21

222318

1413

12

1110

3

4

56

78

9

1a: R= CH3; R'= H : (21R)-argatroban

1b: R= H; R'= CH3 : (21S)-argatroban

Results and Discussion

The complete 1H and 13C NMR assignments (Table 1) forcompounds 1a and 1b were achieved using a combinationof 1D and 2D (COSY, HSQC, HMBC and NOESY) experiments,recorded at 298 K in CD3OD. As an unambiguous entry point tothe complete argatroban resonance assignment, the characteristicH-16 aromatic doublet of doublet was used. This signal was splitinto two resonances that were centered at 6.54 and 6.53 ppm andwere related to the 1a and 1b diastereomer, respectively. Theassignment of these signals was based on their relative ratio ofthe argatroban applied as drug as a 64/36 mixture of 1a and 1b

∗ Correspondence to: Diego Colombo, Dipartimento di Chimica, Biochimica eBiotecnologie per la Medicina, Universita di Milano, Via Saldini 50, 20133Milano, Italy. E-mail: diego.colombo@unimi.it

a Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina,Universita di Milano, Via Saldini 50, 20133 Milano, Italy

b Poli Industria Chimica s.p.a., via Volturno 41, 20089 Rozzano (MI), Italy

c Dipartimento di Chimica Organica, Universita di Pavia, Via Taramelli 10, 27100Pavia, Italy

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(see ‘Experimental’).[5,8] Other 1H and 13C resonances of 1a and1b were assigned in this way when possible (see Table 1). NOESYcross-peaks between the 7.07 ppm aromatic proton and twosignals at about 2.8 and 2.4 ppm allowed us to assign the aromaticH-17 (and consequently the H-15) and the two H-22 resonances.The other tetrahydroquinoline protons (two methylenic H-20and one methine H-21 protons) and the 23-methyl group weresubsequently assigned by COSY correlations (Table 1).

As the second 13-methyl doublet at 0.85 ppm showed a COSYcross-peak with H-9 which was overlapped to other resonances(multiplet at 1.41–1.56 ppm, see Table 1), the piperidine moietyprotons assignment was not possible starting from this correlation.However, it was possible to assign the other two methine protonsof the molecule (H-7 and H-5) from the combined HSQC and COSYspectra. In particular, the piperidine H-7 was distinguished from thearginine H-5 proton because of their different correlation patterns.In fact, the methine signal at 4.05–4.15 ppm was related to two

methylene protons at 0.89 and 2.14 ppm, which in turn showedonly a cross-peak with the H-9 proton, allowing the assignmentof these resonances to H-7 (4.05–4.15 ppm), H-8a (0.89 ppm) andH-8b (2.14 ppm), respectively. Also, the HMBC spectrum confirmedthe H-8 assignments because of a long-range coupling betweenC-13 and H-8a (see Table 1). The remaining methine resonance at3.96 ppm was assigned to H-5, and the complete assignment ofall piperidine and arginine protons of argatroban was obtainedon the basis of COSY and HMBC spectra, the observed C-13/H-10aand C-1/H-2a and H-2b long-range couplings being particularlyuseful for this purpose (Table 1).

The assignments of the geminal protons H-8, H-10, H-11,H-20 and H-22 of both 1a and 1b were accomplished bycomparison of the experimental vicinal coupling constants withthose calculated with the Altona equation[9] as well as with theRamsey’s nonrelativistic approach[10] (Table 1) on the optimizedconformations of compounds modeled with the arginine side

Table 1. 1H, 13C-NMR data, and HMBC correlations of 1a and 1b

δH (ppm) δH (ppm)Measured

J (Hz)Estimated vicinal

J (Hz)aEstimated vicinal

J (Hz)b δC (ppm) δC (ppm) HMBC (H → C)

H/C 1a 1b 1a and 1b 1a 1a 1a 1b 1a and 1b

1 157.2 157.2

2a 3.05 3.07 13.4, 6.2, 6.2 40.0 40.0 C-1, C-3, C-4

2b 3.13 3.14 13.4, 6.2, 6.2 C-1, C-3, C-4

3a 1.61–1.73 1.61–1.73 nd 23.5 23.5 C-2c, C-5d

3b 1.61–1.73 1.61–1.73 nd C-2c, C-5d

4a 1.41–1.56 1.41–1.56 nd 29.1 29.2 C-2e, C-3, C-5, C-6

4b 1.61–1.73 1.61–1.73 nd C-2c, C-3, C-5d, C-6

5 3.96 3.98 14.0, 3.4 (2.6)f 51.7 51.7 C-3, C-4, C-6

6 169.9 169.9

7 4.05–4.15 4.05–4.15 nd 5.1, 1.9 6.2, 2.1 58.3 58.2 C-6

8a (ax) 0.89 0.86 13.0, 13.0, 5.9 12.3, 5.1 10.5, 6.2 36.1 36.1 C-7, C-9, C-10, C-12, C-13

8b (eq) 2.14 2.12 13.0, 5.4, 2.4 3.5, 1.9 3.4, 2.1

9 1.41–1.56 1.41–1.56 nd 12.3, 12.3, 3.5, 3.4 10.5, 9.8, 3.4, 3.4 27.4 27.4 C-11e

10a (ax) 0.64 0.60 13.0, 13.0, 13.0, 4.3 12.4, 12.3, 4.4 11.2, 9.8, 4.6 32.8 32.7 C-8, C-9, C-13

10b (eq) 1.41–1.56 1.41–1.56 nd 3.5, 3.4, 2.1 3.4, 3.0, 2.3 C-11e

11a (ax) 2.85 2.85 13.3, 13.0, 3.0 12.4, 3.5 11.2, 3.0 39.7 39.7 C-6, C-9, C-10

11b (eq) 4.05–4.15 4.05–4.15 nd 4.4, 2.1 4.6, 2.3 C-6

12 175.9 175.9

13 0.85 0.85 6.3 20.9 20.9 C-8, C-9, C-10,

14 118.2 118.0

15 7.42 7.42 7.6 127.5 127.5 C-17, C-18, C19

16 6.54 6.53 7.6, 7.6 114.3 114.1 C-14, C-15, C-17, C-18, C-19

17 7.07 7.07 7.6 133.9 133.7 C-15, C-19, C22

18 122.8 123.1

19 142.9 142.8

20a (ax) 2.96 2.62 11.5, 9.2 10.4 8.1 47.7 47.7 C-23, C-21, C-22, C-19

20b (eq) 3.39 3.45 11.5, 4.1, 2.2 3.9 3.8 C-21, C-19

21 1.92–2.02 1.92–2.02 nd 11.9, 10.4, 3.9, 2.8 9.0, 8.1, 4.1, 3.8 25.6 25.6

22a (ax) 2.39 2.42 16.2, 10.1 11.9 9.0 35.6 35.7 C-17, C-19, C-20, C-21

22b (eq) 2.83 2.74 16.2, 4.0, 2.2 2.8 4.1 C-17, C-19, C-20, C-21

23 1.04 1.05 6.3 17.6 17.5 C-21, C-22, C-18

a Values estimated with the electronegativity-modified Karplus relationship.b Values estimated with the Ramsey’s nonrelativistic approach.c,d,e Overlapped cross peaks.f 1b. Axial (ax) and equatorial (eq) protons are referred to the calculated preferred conformations (Fig. 1).nd: not detected due to overlapping.

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NMR analysis of Argatroban

chain shortened to a methyl group (Fig. 1). Moreover, an observedlong-range coupling (J = 2.2 Hz, see Table 1) due to a ‘W-effect’between H-20b and H-22b confirmed their equatorial orientation

Figure 1. 3D plot of the preferred conformation of compound 1a with thearginine side chain shortened to a methyl group.

in the reduced ring of tetrahydroquinoline, and also H-20a/H-22a and H-8a/H-10a NOESY cross-peaks supported the axialorientation of these protons. Finally, the full assignment of allthe 13C resonances was achieved by HSQC and HMBC spectra,the quaternary carbons being assigned by the following HMBCcorrelations: C-1/H-2, C-6/H-4, H-5, H-7 and H-11, C-12/H-8, C-14/H-16, C-18/H-15, H-16, C-19/H-15, H-16, H-17, H-20, H-22 (Table 1).

Interestingly, the H-20b protons of both 1a and 1b werecompletely separated and well resolved in the used experimentalconditions (3.39 and 3.45 ppm respectively, see Table 1 andFig. 2), and their careful integration could be employed for themeasurement of the diastereomeric composition of argatrobaninstead of using HPLC methods.[5]

Experimental

General

Argatroban was a generous gift from Poli Industria Chimica s.p.a.Rozzano (MI) – Italy. The 64/34 (21R/21S) diastereomeric ratiowas determined by HPLC analysis using an Agilent 1100 DADinstrument equipped with a Zorbax SB C18 column (100×4.6 mmi.d., 1.8 µm particle, Agilent), eluting with a 25/75 (v/v) mixture ofmethanol/acetonitrile (1/1, v/v) and aqueous ammonium acetate(1 g/l) as the mobile phase, at a flow of 1.3 ml/min at 70 ◦C.A solution (5 µl) of the sample (10 mg) in methanol (1 ml) wasinjected and the peaks were detected at 260 nm.

NMR spectroscopy

All NMR spectra were recorded at 298 K with a Bruker AVANCE-500spectrometer operating at 500.13 and 125.76 MHz for 1H and 13C,respectively, using a 5-mm z-PFG (pulsed field gradient) broadband

Figure 2. H-20b protons of 1a and 1b and their integration.

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reverse probe. Chemical shifts are reported on the δ (ppm) scaleand are relative to residual methanol signals (3.30 for 1H and47.6 ppm, central line, for 13C spectra, respectively), and scalarcoupling constants are reported in hertz. The data were collectedand processed by XWIN-NMR software (Bruker) running on a PCwith Microsoft Windows XP. Argatroban, as a mixture of isomers1a and 1b (10 mg), was dissolved in CD3OD (0.6 ml) and insertedunder N2 into a 5-mm NMR tube. The signals assignment was givenby a combination of 1D and 2D COSY, HSQC, HMBC and NOESYexperiments, using standard Bruker pulse programs. The 1H–1Hbond correlations were confirmed by COSY experiment. Theprotonated carbon positions were confirmed by HSQC experiment.The quaternary carbons were confirmed by HMBC experiment. Z-PFGs were used to obtain 1H–1H COSY, HSQC and HMBC spectra.The pulse widths were 7.15 (90◦) and 13.0 µs (90◦) for 1H and 13C,respectively. Typically, 32 K data points were collected for one-dimensional spectra. Spectral widths were 11.45 ppm (5733 Hz)for 1H NMR (digital resolution: 0.17 Hz per point) and 259.84 ppm(32 680 Hz) for 13C NMR (digital resolution: 1.0 Hz per point), 1.5 Hzline broadening. 2D experiments parameters were as follows. For1H–1H correlations: relaxation delay 2.0 s, data matrix 1 K × 1 K(512 experiments to 1 K zero-filling in F1, 1K in F2), 2 or 24transients in each experiment for COSY and NOESY respectively,spectral width 7.7 ppm (3858.0 Hz). A sinebell weighting wasapplied to each dimension. The NOESY spectra were generatedwith a mixing time of 1.0 s and acquired in the TPPI mode.There were not significant differences in the results obtained atdifferent mixing times (0.5–1.5 s). For 13C–1H correlations (HSQCand HMBC): relaxation delay 2.5 s, data matrix 1 K × 1 K (512experiments to 1K zero-filling in F1, 1K in F2), 6 transients ineach experiment, spectral width 7.7 ppm (3858.0 Hz) in the protondomain and 180.0 ppm (22 638.6 Hz) in the carbon domain. Asinebell weighting was applied to both 1H and 13C dimensions.In the HMBC experiments, the delays were set to 3.45 ms (1/(22J)C, H) and 166.7 ms (corresponding to an average 1/(2 nJ)C, H)of 3.0 Hz). All 2D spectra were processed with the Bruker softwarepackage.

Calculations

Calculations were carried out using the GAUSSIAN03 programpackage.[11] The significant conformations of compounds, withthe arginine side chain shortened to a methyl group, were

optimized in the gas phase at the B3LYP/6-311+G(2df,p) levelfor S and 6-311+G(d,p) level for the other atoms to correctlydescribe compounds that contain a sulfur atom and includingdiffuse functions, important for molecules with lone pairs.

Acknowledgements

This study was supported by the University of Milan (Italy) and theUniversity of Pavia (Italy). We thank Professor Fiamma Ronchettiand Professor Lucio Toma for helpful discussions.

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www.interscience.wiley.com/journal/mrc Copyright c© 2008 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2008; 46: 99–102