NMR spectroscopic analysis of new spiro-piperidylrifamycins

MAGNETIC RESONANCE IN CHEMISTRYMagn. Reson. Chem. 2005; 43: 269–282Published online 27 January 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1545

NMR spectroscopic analysis of newspiro-piperidylrifamycins

Eduardo Rubio,1∗ Isabel Merino,1 Ana-Belen Garcıa,1 Marıa-Paz Cabal,1 Cristina Ribas2

and Miguel Bayod-Jasanada2

1 Instituto Universitario de Quımica Organometalica ‘Enrique Moles’, Unidad Asociada al CSIC, Universidad de Oviedo, C/Julian Claverıa 8, 33006Oviedo, Spain2 Asturpharma SA, Polıgono Industrial Silvota, 33192 Silvota, Asturias, Spain

Received 4 October 2004; Accepted 25 November 2004

New spiro-piperidylrifamycin derivatives are presented. These compounds were synthesized fromthe reaction of 3-amino-4-iminorifamycin S and enantiomerically pure 4-piperidones, which generatediasteroisomeric rifabutin analogues with a new stereocentre at the spiranic carbon. The 1H and 13C NMRspectra of these new compounds, and also the configuration of the new stereogenic centres, were assignedusing 2D NMR spectroscopic techniques. A preliminary study of the 1H and 13C NMR spectra of thestarting compounds rifamycin S, 3-amino-4-iminorifamycin S and the related rifabutin was also carriedout and as a result, their previously published 13C NMR data were corrected. Copyright 2005 John Wiley& Sons, Ltd.

KEYWORDS: NMR; 1H NMR; 13C NMR; gHMBC; gCOSY; gNOESY; ROESY; rifamycin S; 3-amino-4-iminorifamycin S;rifabutin

INTRODUCTION

Rifamycins are a family of naphthalenic ansamycin antibio-tics1 isolated from N. mediterranei. They are generallyactive against Gram-positive bacteria and mycobacteria,especially M. tuberculosis, and also possess some activityagainst bacterial DNA-dependent RNA polymerases.2 Thestructure of the rifamycins was determined by chemicaldegradation of rifamycin S (1, Scheme 1), NMR spectroscopy(for some examples, see Ref. 3) and x-ray crystallographicstudies.4 These large macrolide antibiotics are characterizedby having a 17-membered aliphatic bridge, called ansa,connecting two non-adjacent positions on a chromophoricnaphthahydroquinone nucleus and closed by an amidelinkage.

Spiro-piperidyl substituted rifamycin fabbreviated namefor 3-amino-4-deoxo-4-imino-3,4-imidazolinyl-(1H)-[2-spiro(4-piperidyl)]rifamycin Sg derivatives are a class of semisyn-thetic rifamycin antibiotics5 in which the C-3 and C-4positions have been incorporated into an imidazolyl ringbearing a spiro-piperidyl group. The nitrogen of the piperidylring can be substituted with linear and branched aliphaticchains.6 Among them, rifabutin7 (2, Scheme 1) has been

ŁCorrespondence to: Eduardo Rubio, Instituto Universitario deQuımica Organometalica ‘Enrique Moles’, Unidad Asociada alCSIC, Universidad de Oviedo, C/Julian Claverıa 8, 33006 Oviedo,Spain. E-mail: [email protected]/grant sponsor: Asturpharma SA.Contract/grant sponsor: Consejerıa de Educacion y Cultura delPrincipado de Asturias (FICYT); Contract/grant number:PC-CIS01-22; PR-01-GE-9.

introduced in therapeutics as a resistant tuberculosis multi-drug and against MAC (Mycobacterium avium–Mycobacteriumintracellulare complex) infections in AIDS patients.

The synthesis of these spiro-piperidylrifamycin deriva-tives (5) can be carried out by a condensation reactionbetween the common intermediate 3-amino-4-iminorifamy-cin S (3) (obtained from rifamycin S according to differ-ent procedures;8 see also Ref. 5b) and N-substituted-4-piperidones (4), as shown in the Scheme 2, leading to theformation of a new stereocentre at the spiranic carbon atomC-10. A research theme in our laboratory for many yearshas been the stereoselective synthesis of 4-piperidones bythe imino-Diels–Alder reaction of 2-amino-1,3-butadieneswith imines.9 With this issue in mind we decided to utilizeour methodology towards the preparation of new rifabutinanalogues for further biological evaluation. Moreover, thenew analogues prepared in this way will constitute the firstexamples in which enantiomerically pure 4-piperidones willbe used for the condensation reaction with the intermediate3-amino-4-iminorifamycin S (3).

The condensation reaction of (3) was carried out withthe meso-N-allyl-2,6-bis(2-iodophenyl)-4-piperidone (4a)10

and with the enantiomerically pure trisubstituted 4-piperidones (�)-(2R,3S,6S)-2-hydroxymethyl-3-methyl-6-p-methoxyphenyl-4-piperidone (4b)11 and (�)-(2R,3S,6S)-2-hydroxymethyl-3-methyl-6-phenyl-4-piperidone (4c).11 Inthe three cases, new spiro-piperidylrifamycins (ES patentapplication number P200401767/8) were obtained as adiastereoisomeric mixture in the ratio 5a (76 : 24), 5b (90 : 10)and 5c (90 : 10) as determined by high-performance liq-uid chromatography (HPLC). These diasteroisomers were

Copyright 2005 John Wiley & Sons, Ltd.

270 E. Rubio et al.

Scheme 1. Structures of rifamycin S (1) and rifabutin (2).

Scheme 2. Retrosynthetic pathway for the synthesis of rifabutin analogues.

separated and isolated by preparative thin-layer chro-matography (TLC): 5a,b,cM (major) and 5a,b,cm (minor)(Scheme 3).

In this paper we report the structural characterization ofthe new rifabutin analogues 5a,b,cM and 5a,b,cm (Scheme 3)based mainly on their gHSQC and gHMBC spectra. Wealso establish the spatial disposition of the piperidonefragment relative to the rifamycin core in the major andalso in the minor diastereoisomers. This stereochemicalcharacterization was based on their gNOESY and ROESYspectra. We also report the 1H NMR assignment of theintermediate 3-amino-4-iminorifamycin S (3), since so faras we are aware, only 13C NMR data have been publishedfor this compound. The previously assigned 1H and 13CNMR spectra of rifamycin S,12 rifabutin13 and 3-amino-4-iminorifamycin S5b were included as references and wherenecessary were revised as a result of these investigations.

Additionally, this work will contribute to further studieson the structure and conformational properties of relatedmolecules.

RESULTS AND DISCUSSION

The 1H and 13C NMR spectra of the rifamycins have beenstudied in detail by the groups of Gallo,12b – d Whitlock12e,14

and Segre4b in their investigations of the relationship betweenthe solution conformation of this family of compounds andtheir biological activity. Exhaustive 1H selective decou-pling was employed to establish the assignment of the13C NMR spectrum of rifamycin S,12d while its 1H NMRassignment4b,12c,e was based on selective irradiation exper-iments, D2O exchange and approximate chemical shift cal-culations. Recently, a new NMR study of rifamycin S12f andrifabutin13b appeared in which modern 2D NMR techniques

Copyright 2005 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2005; 43: 269–282

NMR analysis of new spiro-piperidylrifamycins 271

Scheme 3. Structures of rifabutin analogues 5a,b,cM and 5a,b,cm.

were used for the assignment and where the previously pub-lished 1H NMR and 13C NMR data were corrected. We firstanalysed the 1H and 13C NMR spectra of the starting materi-als rifamycin S, rifabutin and the 3-amino-4-iminorifamycinS intermediate to use them as references for the assignment ofthe rifamycin skeleton signals in our new rifabutin analogues.After an initial survey of deuterated solvents, we chose C6D6

for the NMR study of these compounds, since it gave clearerand less overcrowded spectra than CDCl3. For rifamycin Swe obtained a set of 1H NMR data that were in agreementwith that reported by Santos et al.12f but the 13C NMR assign-ment we obtained matched the data reported previously by

Martinelli et al.12d The controversy lies in the attributions ofthe signals belonging to the quaternary carbons of the naph-thohydroquinone group. Also, some disagreement with thereported 13C NMR data of the naphthohydroquinone groupwas found when the 13C NMR spectra of rifabutin13b and3-amino-4-iminorifamycin S5b were analysed.

Rifamycin S and 3-amino-4-iminorifamycin S andrifabutinRifamycin S (1)In the 1H NMR spectrum of rifamycin S, the olefinic protonsH-17, H-18, H-19, H-28 and H-29 are easily identified throughtheir chemical shifts, multiplicities and scalar coupling


272 E. Rubio et al.

constants. The strategy of work started by assigning theseolefinic signals in the gCOSY spectrum and following theirconnections through the ansa chain. Second, the analysis ofa deuterated sample of the compound led to the locationof the D2O-exchangeable protons OH-8, NH-2, OH-21 andOH-23. In the next step, the combined use of the gHSQC andgHMBC correlation maps led to the attribution of the signalsat the 1H and 13C NMR spectra to the protons and carbons ofthe molecule (see Table 1). The assignment of the quaternary

carbons of the naphthohydroquinone group was achievedfrom the examination of the correlations of the protonsof the substituents Me-14, OH-8, H-3 and NH-2 with thecarbon nuclei of the chromophore in the gHMBC spectrumand corroborated from the analysis of the 1,1-ADEQUATEspectrum. This last part of the 13C NMR assignment isdescribed next in detail.

The columns for rifamycin S (1) in Table 2 summarizethe long-range correlations of the substituents of the

Table 1. Data for rifamycin S (1) and 3-amino-4-deoxo-4-iminorifamycin S (3)

Rifamycin S (1) 3-Amino-4-deoxo-4-iminorifamycin S (3)

Site

13C NMR(ppm)

1H NMR(ppm) Multiplicity J (Hz)

13C NMR(ppm)


1 185.4 183.22 139.9 111.0NH-2 8.49 s 8.49 s3 117.1 8.24 s 145.3NH2-3 6.94, 7.074 182.0 157.3NH-4 14.78 s5 111.7 109.36 172.8 171.87 115.4 113.58 167.1 169.3OH-8 12.78 s 15.27 s9 111.4 110.010 131.4 129.811 191.1 196.812 109.1 107.413 22.7 1.81 s 21.3 1.75 s14 7.7 2.45 s 7.3 2.38 s15 169.5 168.416 131.4 132.217 133.8 6.06 d 8.8 132.0 6.10 d 11.018 124.8 6.41 dd 15.6, 7.3 124.8 6.71 dd 15.9, 10.519 142.8 5.90 dd 15.6, 7.3 140.9 6.14 dd 15.8, 7.120 40.1 2.34 m 38.0 2.47 m21 74.1 3.61 d 9.6 73.8 3.96 d 10.0OH-21 3.75 s 3.75 s22 33.3 1.72 m 33.6 1.80 m23 77.9 3.08 bd 9.4 77.1 3.17 bd 7.6OH-23 4.05 d 3.4 4.19 d 5.424 37.6 1.60 m 37.7 1.8425 73.9 4.79 d 10.4 73.7 5.23 d 10.626 37.8 1.98 m 38.5 1.62 m27 82.4 3.24 dd 8.0, 2.7 79.1 3.32 dd 6.4, 1.128 115.8 5.35 dd 12.5, 8.2 118.0 5.42 dd 12.5, 6.529 145.8 6.42 d 12.7 142.9 6.32 dd 12.5, 1.130 20.5 1.99 s 20.2 2.05 d 0.831 16.8 0.79 d 6.8 17.4 0.88 d 7.032 11.9 1.06 d 6.9 11.3 1.19 d 7.033 9.1 0.59 d 6.8 8.7 0.66 d 6.934 12.3 0.30 d 7.1 10.2 0.23 d 7.035 173.5 171.636 20.9 1.76 s 20.2 1.70 s37 56.6 2.95 s 56.7 2.91 s



Table 2. Selected cross peaks in gHMBC spectra of 1, 2 and 3

Rifamycin S (1) Rifabutin (2) 3-Amino-4-deoxo-4-iminorifamycin S (3)

Site OH-8 NH-2a H-3 Me-14 OH-8 NH-2a Me-14 OH-8 NH-4 NH-2a Me-14

C-1 4J�C,H�b 3J�C,H� 3J�C,H� 3J�C,H� 3J�C,H�

C-2 2J�C,H� 2J�C,H�

C-3 3J�C,H� 3J�C,H� 3J�C,H� 3J�C,H�

C-4 4J�C,H� 6J�C,H� 2J�C,H�

C-5 4J�C,H� 4J�C,H� 4J�C,H�

C-6 4J�C,H� 3J�C,H� 4J�C,H� 3J�C,H� 3J�C,H�

C-7 3J�C,H� 2J�C,H� 3J�C,H� 2J�C,H� 3J�C,H� 2J�C,H�



C-10 3J�C,H� 5J�C,H� 5J�C,H� 3J�C,H� 5J�C,H�

a NH-2 shows another cross peak with C-15.b This cross peak could also be considered a 2J�C,H� through an H-bond.

NH−23OH−8

11

14

356

815

2919

2

1716

18

3 287 5

12

10

9

34333132

2422

3613

30

2620

1437

OH−8/9

18 1929

17 2528OH−23

OH−21

2127

23

OH−8/7

OH−8/6

OH−8/8

OH−8/1NH−2/4NH−2/1 3/1

3/2

3/5 14/5

14/9

14/7

14/10

14/8

14/6NH−2/15

NH−2/3

3/10

ppm

14 13 12 11 10 9 8 7 6 5 4 3 2 1 ppm

110

120

130

140

150

160

170

180

190

Figure 1. Detail of the gHMBC spectrum of 1.

naphtohydroquinone group OH-8, NH-2, H-3 and Me-14with the carbon atoms of the chromophore (see also thegHMBC correlation map of 1 depicted in Fig. 1). The 1HNMR signal of OH-8 at υ 12.73 ppm correlates with fivecarbons at υ 111.4, 115.4, 167.1, 172.8 and 185.4 (a low-intensity cross peak) ppm. The peaks at υ 115.4, 167.1 and172.8 ppm show additional intense correlations with Me-14(υ 2.37 ppm) and, on account of their chemical shifts, canbe assigned to C-7 [3J(C-7,OH-8)], C-8 [2J(C-8,OH-8)] and

C-6 [4J(C-6,OH-8)] respectively. Obviously, the cross peakat υ 111.4 ppm represents the three-bond connectivity ofOH-8 with C-9. On the other hand, the carbon atom at υ131.4 ppm shows a strong cross peak with H-3 and anotherweak correlation with Me-14 and it should be assigned toC-10 [5J(C-10,Me-14) and 3J(C-10,H-3)]. The assignment ofC-2 is derived from locating the HMBC cross peak of H-3with the carbon at υ 139.9 ppm [2J(C,H)]. The distinctionbetween the two carbonyl groups was possible through the


274 E. Rubio et al.

cross peak of the proton atom OH-8 and the carbon signalat υ 185.4 ppm, which would correspond therefore to C-1 [4J(C,H)]. At this point, the only quaternary carbon notassigned is the peak at υ 111.7 ppm. A correlation of thiscarbon with H-3 via 4J(C,H) and with Me-14 via 4J(C,H)confirms its assignment for C-5 and completes the 13C NMRassignment of the naphthohydroquinone group.

Additional confirmation for these assignments comefrom the 1,1-ADEQUATE 2D NMR experiment15 that allowsone to discriminate cross peaks arising from 2J(C,H) and3J(C,H) in the gHMBC spectrum.16 Thus, C-2 and C-4were unambiguously assigned by the cross peaks of H-3 with υ 139.9 ppm and υ 182.0 ppm, respectively. Thesearguments lead to the conclusion that the 13C NMR datareported by Santos et al.12f for C-1, C-2, C-4, C-5 and C-10 are erroneous (the assignments for C-1 and C-4 shouldbe interchanged and those for C-2, C-5 and C-10 shouldalso be interchanged) and agree with the results publishedby Martinelli et al. in 1974.12d Although the NMR data wereport here were measured in C6D6 and the data publishedby Santos et al. were obtained in CDCl3, the values of thechemical shifts correlate in both solvents. To confirm ourstudy, we performed NMR measurements of 1 in CDCl3

and the analysis of the gHSQC and the gHMBC spectrumled to the same 13C NMR assignment as we had obtainedpreviously for 1 in C6D6. On the other hand, the 1H NMRassignment for Me-13 and Me-36 in C6D6 published by Caseyand Whitlock12e in 1975 should be also exchanged.

3-Amino-4-iminorifamycin S (3)In the case of 3, the assignment of the 1H and 13C NMRspectra (see Table 1) was carried out after deuteration tolocate the labile NH-2, NH2-3, NH-4, OH-8, OH-21 and OH-23 protons and from the analysis of the gCOSY, gHSQCand gHMBC spectra. First, from the analysis of the gCOSYspectra, the characteristic olefinic protons H-17, H-18, H-19,H-28 and H-29 were assigned followed by the location ofthe protons in the ansa chain linked to the olefinic hinges,H-20 and H-27. From these protons, the connections withH-21, Me-31 and Me-30 were established and, therefore,the corresponding carbons could be identified by readingthe gHSQC spectrum. Second, the combined analysis of thelong-range correlations in the gHMBC spectrum and thegHSQC spectrum led to the full assignment of the carbonatoms in the ansa chain and also to the assignment of all thesubstituents bonded to the ansa chain (see Table 1).

The 13C NMR assignment of the naphthohydroquinonegroup was deduced from the analysis of the HMBC crosspeaks of the substituents OH-8, NH-4, NH-2 and Me-14 (the3-amino group did not show any correlation peak in theHMBC spectrum). Table 2 gives a summary of the observedcorrelations between these protons and the carbon atomsof the chromophore. The 1H NMR signal of OH-8 at υ15.27 ppm correlates with three carbons at υ 110.0, 113.5and 169.3 ppm. The chemical shift identifies the downfieldcarbon at υ 169.3 ppm as C-8, whereas the two other crosspeaks represent the 3J(C,H) correlations of OH-8 with C-9and C-7. These carbons can be assigned and discriminatedas C-7 (υ 113.5 ppm) and C-9 (υ 110.0 ppm) on account of

the different intensities of their cross peaks with Me-14 via2J(C,H) (strong cross peak) and via 4J(C,H) (weak cross peak),respectively, and by comparison with their chemical shifts in1. On the other hand, the 1H NMR signal of the iminic NH-4at 14.78 ppm show three intense cross peaks at υ 129.8, 145.3and 157.3 ppm and a weak one at υ 111.0 ppm. Chemical shiftconsiderations, together with the three-bond connectivity ofthe NH-2 proton atom with the signal at υ 145.3 ppm, allowthe assignment of C-10 [υ 129.8, 3J(C-10,NH-4) correlation],C-3 [υ 145.3, 3J(C-3,NH-4) correlation] and C-4 [υ 157.3,2J(C-4,NH-4) correlation]. The two quaternary carbon atomsremaining at υ 111.0 and 109.3 ppm were tentatively assignedto C-2 and C-5 based on their chemical shifts in rifamycinS (1) and rifabutin (2) that will be discussed next. Hence thepreviously published5b 13C NMR assignments for 3 in C6D6

at 20 MHz have been revised and rectified.

Rifabutin (2)In the same way, as described above for 1 and 3, the 1H and13C NMR assignments of the rifabutin (2) was accomplished(see Table 3). Only the controversial 13C NMR assignmentsof the naphthohydroquinone group will be discussed indetail. Table 2 gives a summary of the correlations of thesubstituents at the naphthohydroquinone group OH-8, NH-2 and Me-14 with the carbon atoms of the chromophore(the 3-amino group did not show any correlation peak inthe HMBC spectrum). The carbon atoms C-6, C-7, C-8 andC-9 were assigned to υ 171.4, 114.4, 168.4 and 112.0 ppmby analysing the cross peaks of these carbons with theOH-8 and Me-14 groups as previously discussed for the3-amino-4-iminorifamycin derivative (3). On the other hand,the proton NH-2 showed three intense cross peaks at υ181.8, 168.1 142.4 ppm and a fourth weak cross peak at υ105.3 ppm. On account of their chemical shifts, these peakscan be attributed to C-1 [3J(C,H)], C-15 [2J(C,H)], C-3 [3J(C,H)]and C-2 [2J(C,H)], respectively. The assignment of C-3 at υ142.4 ppm is in good agreement to the well established C-3 inthe 3-amino-4-iminorifamycin S derivative (3) (υ 145.3 ppm)discussed above. The carbon atoms C-5 and C-10 can bedistinguished in terms of expected chemical shift. Hence thesignal at υ 109.5 ppm belongs to C-5 and its assignmentis confirmed by a small cross peak of C-5 with Me-14via 4J(C,H). The remaining signal at υ 125.4 ppm shouldcorrespond to C-10. It is noteworthy how the planarityof the system favours long range correlations [4J(C,H),5J(C,H) and 6J(C,H)] of Me-14 with all the carbon atoms ofthe naphthohydroquinone group (see Table 2). The upfieldlocation of C-5 with regard to C-10 is supported by thechemical shift of these atoms in compounds 1 (see Table 1)and 3 (see Table 1), and also by the 13C NMR data forrifampicin,17 another biologically active compound of therifamycin family. To be able to carry out a comparison withthe 13C NMR data of rifabutin previously reported13b inCDCl3, we performed the NMR measurements of 2 in thissolvent and we found that the analyses of the gHSQC andthe gHMBC spectra led to the same 13C NMR assignmentas previously obtained in C6D6. As a conclusion of theseanalyses, the published assignments for C-2 and C-3 shouldbe interchanged, and also those for C-5 and C-10.



Table 3. Data for rifabutin (2)

Site

13C NMR(ppm)


1 181.82 105.3NH-2 8.63 s3 142.4NH-3 9.42 s4 155.35 109.56 171.47 114.48 168.4OH-8 15.40 s9 112.010 125.411 191.512 107.713 21.8 1.80 s14 7.6 2.58 s15 168.116 131.717 132.5 6.08 d 9.818 124.4 6.61 dd 15.8, 9.919 140.9 5.98 dd 15.8, 7.020 38.8 2.45 m21 72.8 3.86 d 9.7OH-21 3.70 bs22 33.5 1.77 m23 77.0 3.20 d 10.3OH-23 4.05 bs24 37.8 1.7125 73.2 5.08 d 10.626 38.2 1.8827 81.1 3.24 dd 7.8, 2.628 115.7 5.48 dd 12.6, 7.829 144.9 6.42 d 12.630 20.3 2.03 s31 17.1 0.82 d 6.932 11.7 1.19 d 7.033 8.9 0.71 d 6.834 11.2 0.26 d 7.135 171.736 20.3 1.74 s37 56.2 2.92 s10 94.720a 36.4 1.88(ax) m

2.03(eq) m30b 51.5 3.11(ax) m

2.60(eq) m40b 51.3 2.97(ax) m

2.60(eq) m50a 35.7 1.88(ax) m

2.03(eq) m60 66.3 2.15 d 7.070 25.8 1.73 m80, 90 20.7 1.027,1.022 d, d 6.4, 6.4

a Sites 20 and 50 are indiscernible, so might be interchanged.b Sites 30 and 40 are indiscernible, so might be interchanged.

A careful study of the DEPT, gHSQC and gCOSY spectraallowed the assignment of all the signals of the newlyincorporated piperidyl moeity in the 1H NMR spectrum (seeTable 3). When compared with the 1H NMR data of rifabutinmeasured in CDCl3, in which a broadening of the piperidinesignals is observed, the data set we obtained in C6D6 allowsthe distinction of the diasterotopic protons of a chair-likeconformation with H-30 and H-40 in an axial position at υ 3.11and 2.97 ppm, while their geminal counterparts appear as amultiplet centred at 2.60 ppm. The equatorial H-20 and H-50

appear at a chemical shift of 2.03 ppm overlapped by Me-30,while their axial counterparts appear as a multiplet centredat 1.88 ppm together with H-26.

Spiro-piperidylrifamycins (5)Next, we studied the spiro-piperidylrifamycins (5) (Scheme2) that were obtained as a mixture of diastereosiomers, whichwill be named 5M (major) and 5m (minor) (Scheme 3). Eachpair of diastereoisomers were quantified by HPLC and sep-arated by preparative TLC. The structural assignment of5M and 5m was accomplished through the analysis of theirgCOSY, gHSQC and gHMBC spectra, while the stereochem-ical characterization of the new formed stereogenic centreC-10 was achieved from a study of the gNOESY and 1D-ROEspectra (5aM and 5am) or the ROESY spectra (compounds5b,cM and 5b,cm).

Tables 4 and 5 summarize the structural characterizationof 5aM, 5am and 5bM, 5bm respectively. The 1H and13C NMR assignments for 5cM and 5cm are availableas supplementary material, since there are no significantdifferences with regard to 5bM and 5bm. The discussionfor compounds 5 will be focused on the assignment of thecarbon and proton nuclei of the spiro-piperidyl fragment,with the rest of the molecule presenting no different featureswith regard to the previously discussed 1, 2 and 3.

In 5aM, a clear entry for the analysis of the area ofinterest in the gHMBC spectrum was the iodine supportingcarbons C-100 and C-160 (the more shielded pair of quaternarycarbons) at υ 99.4 and 99.7 ppm, that correlate with a pairof aliphatic protons, easily identified as H-30 and H-40. Theassignment of the methylenic protons H-20 and H-50 wasnot so straightforward, since the two carbons C-20 and C-50

appear at the same chemical shift and, on the other hand,these diastereotopic protons show different chemical shiftsfor the axial and the equatorial orientations and appear ina crowded area (2.00–1.65 ppm) overlapped with Me-30,H-26, H-24, Me-13, Me-36 and H-22. (For clarity, we choseto number the more unshielded signal as H-30. However,since the sites 20, 50 and 30, 40 could not be distinguished,the assignments for both pairs might be interchanged.) Thecoupling pattern of the piperidine protons H-30 [υ 5.48 ppm,dd, 3J�H,H� D 11.2, 3.1 Hz] and H-40 [υ 5.26 ppm, bd,3J�H,H� D 11.1 Hz] agree with an axial orientation of theseprotons and therefore with an equatorial disposition for thelarge aromatic substituents at the piperidine ring. Both H-30

and H-40 show two cross peaks in the gCOSY spectrum withdifferent intensities. The more intense correlations matchthe axial–axial coupling and the less intense correlationthe axial–equatorial coupling, which lead to the location of


276 E. Rubio et al.

Table 4. Data for compounds 5aM and 5am

5aM 5am

Site

13C NMR(ppm)


13C NMR(ppm)


1 182.0 181.92 105.2 106.1NH-2 8.74 s 8.82 s3 141.9 143.1NH-3 8.91 s 10.34 s4 156.0 156.15 109.5 109.46 171.6 171.5 s7 113.9 114.28 168.2 168.5OH-8 15.28 s 15.42 s9 111.8 112.210 125.4 125.4 s11 190.8 191.8 s12 106.8 107.413 21.1 1.77 s 21.5 1.81 s14 7.5 2.49 s 7.5 2.56 s15 168.1 168.316 131.3 131.417 132.7 6.07 d 10.6 132.8 6.02 d 9.918 124.2 6.70 a 124.5 6.70 a

19 141.1 5.99 dd 15.8, 6.7 140.9 5.97 dd 15.8, 7.020 38.4 2.40 m 39.1 2.48 m21 72.6 3.95 d 9.4 72.5 4.04 d 9.3OH-21 3.78 s 3.86 s22 33.5 1.67 m 33.5 1.91 m23 77.1 3.13 dd 8.5, 5.2 77.3 3.19 mOH-23 4.23 d 5.2 4.17 d 4.124 37.6 1.79 m 37.5 1.84 m25 73.8 5.22 d 10.8 73.6 5.09 d 10.426 38.8 1.96 m 38.1 1.56 m27 79.5 3.52 bd 3.9 80.2 3.12 m28 116.5 5.45 dd 12.5, 6.5 115.5 5.32 dd 12.5, 6.829 143.6 6.46 d 12.4 144.1 6.41 d 12.530 20.3 1.99 s 20.6 1.98 s31 17.3 0.75 d 7.0 17.9 0.96 d 6.832 11.5 1.11 d 7.0 11.8 1.20 d 7.033 8.8 0.60 d 6.7 9.4 0.78 d 6.734 10.5 0.30 d 7.0 10.7 0.26 d 7.035 171.8 172.036 20.2 1.73 s 20.2 1.63 s37 56.6 2.96 s 56.4 2.89 s10 93.5 93.920 43.8 1.85(ax) m 42.1 2.79(ax) t 12.6

1.75(eq) m 1.97(eq) m30 65.5 5.48 dd 11.2, 3.1 65.4 4.85 d 10.340 65.4 5.26 bd 11.1 65.6 4.71 d 10.850 43.9 1.93(ax) m 43.3 2.89(ax)

1.80(eq) m 1.93(eq) m60 53.0 3.24 d 6.3 52.3 3.12 m70 132.7 6.79 m 130.9 6.24 m80 119.0 5.38(cis) d 10.5 120.7 5.17(cis) d 10.2

4.85(trans) d 16.9 4.76(trans) d 16.9



Table 4. (Continued)

5aM 5am

Site

13C NMR(ppm)


13C NMR(ppm)


90 146.1 144.5100 99.4 99.6/99.5110, 170 139.7, 139.5 7.75 apparent t 8.5, 9.3 139.8, 139.4 7.76 m120, 180 129.0, 128.9 6.70 a 129.2 6.67 a

130, 190 128.5, 128.4 7.23 m 129.0, 128.7 7.21, 7.15 t, t 7.5, 7.4140 129.6 7.81 d 7.2 129.5/129.3 7.73 m150 145.4 144.3160 99.7 99.6/99.5200 129.2 7.81 d 7.2 129.5/129.3 7.73 m

a Signals for protons 120, 180 and 18 are overlapped.

Table 5. Data for compounds 5bM and 5bm

5bM 5bm

Site

13C NMR(ppm)


13C NMR(ppm)


1 182.2 182.02 105.1 105.3NH-2 8.99 s 8.76 s3 142.9 145.8NH-3 8.72 s 9.47 s4 156.3 156.45 109.3 109.66 171.7 171.67 114.2 114.38 168.5 168.6OH-8 15.52 s 15.45 s9 112.3 112.110 125.7 126a

11 192.2 191.712 107.5 107.513 21.4 1.80 s 21.6 1.78 s14 7.5 2.55 s 7.5 2.55 s15 168.0 168.316 131.2 131.317 133.1 6.12 d 10.0 133.0 6.14 d 10.418 124.3 6.73 dd 15.7, 10.5 124.6 6.73 dd 15.8, 10.119 141.2 6.02 dd 15.8, 7.0 141.2 6.07 dd 15.8, 7.020 38.4 2.46 m 38.3 2.49 m21 72.7 4.03 d 9.5 73.2 3.90 d 9.8OH-21 4.30 bs 3.80 bs22 33.6 1.86 m 33.5 1.80 m23 77.1 3.26 d 10.1 77.2 3.12 mOH-23 4.30 bs 4.14 d 3.924 37.7 1.88 m 37.6 1.77 m25 73.5 5.25 d 10.4 73.4 5.04 d 10.426 38.2 1.76 38.1 1.60 m27 79.5 3.38 d 5.1 80.1 3.15 dd 7.6, 1.828 116.2 5.41 dd 12.6, 6.3 115.8 5.33 dd 12.5, 6.929 143.5 6.41 d 12.6 144.2 6.40 d 12.530 20.5 2.04 s 20.2 2.05 s31 17.4 0.86 d 5.9 17.4 0.90 d 6.9

(continued overleaf )


278 E. Rubio et al.

Table 5. (Continued)

5bM 5bm

Site

13C NMR(ppm)


13C NMR(ppm)


32 11.4 1.22 d 5.8 11.6 1.18 d 7.033 8.9 0.76 d 6.5 8.8 0.55 d 6.934 10.6 0.29 d 6.8 10.8 0.23 d 7.135 171.7 172.036 20.2 1.74 s 20.2 1.64 s37 56.5 2.94 s 56.4 2.88 s10 98.3 98.620 40.2 2.17 bt 38.8 2.8830 60.8 3.96 bd 4.9 61.2 2.8840 57.4 4.75 d 11.5 58.5 3.96 dd 12.0, 2.050 45.5 2.26(ax) apparent t 11.5, 12.2 45.2 3.07(ax) t 12.3

1.64(eq) bs 1.75(eq)60 9.3 0.86 d 5.9 8.8 0.76 d 5.970 64.1 3.75 dd 11.0, 4.6 64.2 3.53 d 3.7

3.68 d 9.990, 130 128.1 7.27 d —a 128.3 7.23 d100, 120 113.9 6.86 d 8.5 113.9 6.87 d 8.6110 159.4 159.5140 54.6 3.44 s 54.6 3.41 s

a Overlapped with solvent.

H-20ax, H-20eq, H-50ax and H-50eq. The use of the gHSQCspectrum helped to establish the final assignment of that areaat the 1H NMR spectrum (see Table 4).

The iodine-supporting carbons C-100 and C-160 were alsothe starting point for the assignment of the aromatic protonsand the rest of the aromatic carbons (Table 4). It was possibleto distinguish C-90, C-100 and C-140 from their partners C-150, C-160 and C-200 on account of the two- and three-bondconnectivities of the first group of carbons with the protonH-30 in the gHMBC spectrum. From the carbons of the otheraromatic ring, only C-160 showed a three-bond correlationwith H-40. However, the pairs of carbons C-110, C-170 andC-120, C-180 and C-130, C-190 could not be discriminated.

The proposed disposition of the substituents (Scheme 4,5aM) is supported by gNOESY cross peaks between theamine proton NH-3 and the protons H-20ax and H-50ax,as can be observed in the detail of the gNOESY spectrumof 5aM depicted in Fig. 2. Additional NOE correlations ofthese protons with the ortho aromatic protons H-140 and H-200, respectively, together with gNOESY cross peaks of theequatorial protons H-20 and H-50 with H-30ax and H-40ax,respectively, lead to the conclusion that the conformation insolution of the spiro-piperidyl unit in this rifabutin analogueis a chair with the large substituents facing up in an equatorialorientation as shown in Scheme 4, 5aM.

In the minor diastereoisomer 5am, the resonances ofthe CH2 carbons C-20 and C-50 were separated enough toprovide an easier assignment of the proton and carbonnuclei of this part of the molecule (see Table 4). Followingthe same procedure as above, the protons H-30 and H-40 wereidentified as two doublets at υ 4.85 [3J�H,H� D 10.3 Hz] andυ 4.71 [3J�H,H� D 10.8 Hz] ppm. With the same numbering

criterion as used above, the more unshielded proton wasattributed to H-30. Both H-30 and H-40 showed only onecorrelation in the gCOSY spectrum and assuming again anaxial disposition for these protons, those cross peaks wouldprovide the assignment of H-20ax and H-50ax. The readingof the gHSQC spectrum gave the correct distinction forcarbons C-20 and C-50 and next, from the cross peaks of thesecarbons, the protons H-20eq and H-50eq could be located.The cis relationship of H-30 and H-40 with H-20eq and H-50eq,respectively, was also revealed by the corresponding crosspeaks observed in the gNOESY spectrum between thesepairs of protons (a detail of the gNOESY spectrum of 5amis shown in Fig. 3). The observed gNOESY cross peaks ofthese axial protons H-20 and H-50 with the ortho aromaticprotons H-140 and H-200, respectively, together with thegNOESY correlations of the amino NH-3 proton with allthe upward-facing substituents (H-30ax, H-40ax, H-20eq andH-50eq) corroborate a chair-like conformation in solution forthis compound, with the aromatic substituents facing downin an equatorial disposition as depicted in Scheme 4, 5am.

The experimental 1D-ROE for 5am and 5aM agreed withthe results obtained from the gNOESY spectra: irradiationof the NH-3 proton resulted in enhancement of the signalsof H-30ax and H-40ax in the minor compound 5am, whereasthese enhancements were not observed in the case of themajor compound 5aM.

The structural assignment of 5bM was easier sincethe protons H-30 and H-40 were readily discriminatedon account of their chemical shifts (see Table 5). Thestrategy of work started by locating the two methyleniccarbons, C-50 (υ 45.5 ppm) and C-70 (υ 64.1 ppm), and theircorresponding protons using a multiplicity-edited gHSQC



Scheme 4

ppm

1.52.02.53.03.54.04.55.05.5 ppm9.5

9.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

14′20′11′17′

13′ 19′7′

291719

283′

4′ 258′c8′t OH−23 21

OH−2127 6′

23 262′ eq5′ eq

24

225′ ax

2′ ax

30 13 361437 32

20

18+12′ 18′

NH−2NH−3

4′/5′ eq

3′/2′ eq

5′ eq/4′

2′ eq/3′

2′ ax/NH−35′ ax/NH−3

5′ ax/20′

28/26

25/24

26/28

24/25

2′ ax/14′

Figure 2. Detail of the gNOESY spectrum of 5aM.


280 E. Rubio et al.

NH−3

NH−2

1917

7′ 29

13′19′

14′20′11′17′

18+12′18′

ppm

2.02.53.03.54.04.55.05.5 ppm11.0

10.5

10.0

9.5

9.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

8′c28 25 3′

8′ t4′ OH−23

21OH−21 23

6′+27

5′ ax

2′ ax 20

37 14 30

222′ eq5′ eq 24

1336

26

3′/2′ eq

4′/5′ eq

3′/NH−3 4′ /NH−3

3′/7′ 4′/7′

2′ eq/NH−35′ eq/NH−3

3′/4′

4′/3′

3′/6′ 4′/6′

5′ ax/20′ 2′ ax/14′

Figure 3. Detail of the gNOESY spectrum of 5am.

technique.18 The CH2-70 protons appeared as a multipletcentred at 3.75 ppm, whereas the diastereotopic CH2-50

protons appeared separated in two different signals for theaxial and the equatorial protons (υ 2.26 ppm, apparent t,J D 11.5, 12.2, and υ 1.64 ppm, bs). Next, the analysis of theircross peaks in the gCOSY spectrum provided the location ofH-40 (υ 4.75 ppm, d, J D 11.5 Hz), H-30 (υ 3.96 ppm, bs) andalso H-20 (υ 2.17 ppm, bt). The magnitude of the couplingconstant of the benzylic proton H-40 [3J�H,H� D 11.5 Hz]agreed with a vicinal axial–axial coupling, which identifiedthe low-field CH2-50 signal at υ 2.26 ppm as the axial H-50 proton, whereas the upfield CH2-50 signal (υ 1.64 ppm)was attributed to the equatorial H-50 proton. The relativedisposition of the substituents (see Scheme 4, 5bM) wasdeduced from the analysis of the ROESY spectrum. TheROESY cross peak of H-40ax with H-50eq, together withthe cross peaks of H-30ax with H-40ax and the methylgroup Me-60, evidence their cis relationship. On the otherhand, the additional ROESY cross peaks of H-50ax with theortho protons H-90 and H-130 of the aromatic ring supporta chair-like conformation in which all the substituentsoccupy an equatorial position and the aromatic ring andthe hydroxymethylgroup are in the upper side of the chairas shown in Scheme 4, 5bM. The spatial proximity of the40-aromatic ring with the rifamycin substituents is supported

by ROESY cross peaks between the aromatic protons H-100,H-120 and the methyl groups of the rifamycin core Me-34 andMe-32.

Following the same strategy as above, the proton andcarbon atoms of the spiro-piperidyl fragment in 5bm wereassigned (see Table 5). The information from DEPT-135together with the gHSQC spectrum led to the location ofthe two methylenic CH2 carbons C-50 (υ 45.2 ppm) and C-70

(υ 64.2 ppm) and to the assignment of the 1H NMR chemicalshift of CH2-70 (d, υ 3.53 ppm) and of the diastereotopicCH2-50 protons, which appeared separated in two differentsignals for the axial and the equatorial protons [υ 3.07,t, 3J�H,H� D 12.3 Hz and 1.75 ppm, overlapped]. Next,the analysis of their cross peaks in the gCOSY spectrumprovided the location of H-40 (υ 3.96 ppm, dd, J D 12.0,2.0 Hz) and H-30 (υ 2.88 ppm, overlapped by Me-37). Thecoupling pattern of H-40 revealed its axial disposition andits coupling with H-50axial with a large coupling constant[3J�Hax,Hax� D 12.0 Hz] and with H-50equatorial with asmall coupling constant [3J�Hax,Heq� D 2.0 Hz], whichpermitted the attribution of the low-field triplet at υ 3.07 ppmfor the H-50axial proton and the upfield overlapped signalat υ 1.75 ppm for the equatorial proton. These assignmentsare supported by ROESY cross peaks of H-40ax with H-50eq and H-30ax, which evidence their cis relationship and



the consequent disposition of the aromatic ring and thehydroxymethyl group in equatorial positions. In addition,the ROESY correlations of the amino NH-3 proton with allthe upward-facing substituents (H-30ax, H-40ax, H-50eq andMe-60) corroborate a chair-like conformation in solution forthis compound, with the aromatic and the hydroxymethylsubstituents facing down in an equatorial disposition asdepicted in Scheme 4, 5bm.

The analysis of the gCOSY and ROESY spectra of 5cMand 5cm resembled the results discussed for 5bM and 5bm,respectively.

In summary, the gNOESY and ROESY cross peaks of theNH-3 amino group with the piperidine protons, in the minorcompounds 5a,b,cm, support a perpendicular disposition ofthe spiro-piperidyl unit in relation to the rifamycin core, sothat the protons H-30 and H-40 would lie on the same side ofthe molecule as the NH-3 amino group. In contrast, in themajor compounds 5a,b,cM, the perpendicular orientation ofthe spiro-piperidyl unit would place H-30 and H-40 on adifferent side of the molecule and the aromatic rings nowclose to the NH-3 amino group, which would account for theshielding that the NH-3 proton experiences in 1H NMR in themajor diastereoisomers related to the minor ones (between0.6 and 1.4 ppm).

Another common feature that makes the major com-pounds different from the minor compounds was derivedfrom the observation of the gCOSY spectra of each pairof diastereosiomers. In the major compounds 5a,b,cM theprotons bonded to carbons bearing a heteroatom (the oddprotons H-21, H-23, H-25 and H-27) showed a characteris-tic coupling pattern. Each of these protons exhibited twocross peaks with the even vicinal protons H-20, H-22, H-24, H-26 and H-28 with different intensity in the followingway: the H-21/H-20, H-23/H-24, H-25/H-26 and H-27/H-28cross peaks are high-intensity signals, whereas the H-21/H-22, H-23/H-22, H-25/H-24 and H-27/H-26 cross peaks arelow-intensity signals. In contrast, in the gCOSY spectra ofthe minor diastereoisomers 5a,b,cm, the low-intensity crosspeaks of these odd protons disappear or become almostimperceptible. This property was observed when the gCOSYspectra were recorded in C6D6 and also when CDCl3 wasused as a solvent (5b and 5c). These changes from themajor to the minor diastereoisomers reflect that the spatialarrangement of the spiro-piperidyl fragment in the moleculehas an influence on the conformation of the ansa chainand therefore on the potential biological activity of each ofthese diastereoisomeric rifabutin analogues, since it is knownthat the biological activity in the rifamycin derivatives is afunction of the spatial disposition of the ansa chain.4b,14

CONCLUSIONS

The 1H NMR spectrum of the 3-amino-4-deoxo-4-imino-rifamycin S has been described and the 13C NMR assignmentof the naphthohydroquinone group in rifamycin S, rifabutinand the 3-amino-4-deoxo-4-iminorifamycin S has beencorrected.

Three spiro-piperidylrifamycin derivatives, which incor-porate a new stereogenic centre in the molecule, were

synthesized as a mixture of two diastereoisomers. The struc-tural characterization of each diastereosiomer was achievedthrough the analysis of their gCOSY, gHSQC and gHMBCspectra, and the configuration of the new spirocyclic C-10

carbon centre was assigned by studying their gNOESY andROESY spectra.

Finally, special features, namely the chemical shift ofthe NH-3 proton and the coupling pattern of the oddprotons of the ansa chain, allow an initial prediction ofthe configuration of the spirocyclic centre in these highlybiologically interesting compounds.

EXPERIMENTAL

Compounds 2,7 38 and the piperidones 4a10 and 4b,c11 wereprepared according to literature procedures. The synthesis ofcompounds 5 and their biological activity will be describedin a separate publication.

NMR spectra were recorded on a Bruker AV 400spectrometer operating at 400.13 and 100.61 MHz for 1Hand 13C, respectively, using a 5 mm QXI 1H/13C/15N/31Preverse probe including a z-gradient coil. Sample amountsranged between 30 and 50 mg, except for spectra of rifamycinS, which were measured from concentrated samples of about150 mg. Deuterated benzene (C6D6) was used as solvent;chemical shifts are reported in ppm and referenced toresidual proton solvent signals (υ 7.16 for proton and υ128.39 for carbon). Typical parameters for 1H NMR spectrawere spectral width 6800 Hz and pulse width 8.5 µs at anattenuation level of 0 dB; line broadening (lb D �0.5 Hz)and a Gauss function (gb D 0.3) were applied prior toFourier transformations of FID to obtain resolution-enhancedspectra. Typical parameters for 13C NMR spectra werespectral width 27 000 Hz, pulse width 9.5 µs at an attenuationlevel of �2 dB and relaxation delay 2 s; WALTZ-16 was usedfor broadband proton decoupling; the FIDS were multipliedby an exponential weighting (lb D 2 Hz) before Fouriertransformation.

All the 2D experiments were acquired and processedusing standard Bruker NMR software. For COSY, gNOESY,HSQC, HMBC, 1,1-ADEQUATE and 1D-ROE experiments,the gradient-enhanced versions of the pulse sequences wereused in order to obtain clean and artefact-free spectra.Gradient selection was achieved through a 5% sine truncatedshaped pulse gradient of 1 ms.

The gCOSY spectra were acquired in the absolutemode. Selected parameters for gCOSY were sweep widthca 6800 Hz, 1024 ð 256 data set, number of scans 4, dataprocessing using zero filling in the F1 domain and shiftedsine-bell apodization of factor 0 in both dimensions. ThegNOESY spectra were acquired in the phase-sensitive mode.Selected parameters for gNOESY were sweep width ca6800 Hz, 2048 ð 256 data set, number of scans 16–24, mixingtime 0.5 s, data processing using zero filling in the F1 domainand shifted squared sine-bell apodization of factor 2 in bothdimensions. The ROESY spectra were acquired in the phase-sensitive mode. Selected parameters for ROESY were sweepwidth ca 6800 Hz, 2048 ð 512 data set, number of scans16–24, power level for the spin-lock pulse 23.88 dB, mixing


282 E. Rubio et al.

time 450 ms, data processing using zero filling in the F1domain and shifted squared sine-bell apodization of factor 2in both dimensions.

The gHSQC spectra were acquired in the phase-sensitivemode with data acquisition of 16 scans and 256 incrementsin F1 and sweep widths in F2 and F1 of ca 3600 and16 200 Hz, respectively. The FIDs were processed using zerofilling in the F1 domain and shifted sine-bell apodization offactor 2 in both dimensions. GARP modulation of 13C wasused for decoupling. The gHMBC spectra were acquired innon-phase-sensitive mode. A low-J filter was incorporatedinto the pulse sequence. Selected parameters for gHMBCwere sweep width ca 6800 Hz for 1H and 21 500 Hz for 13C,1024 ð 512 data set, number of scans 16–32, data processingusing zero filling in the F1 domain and shifted sine-bellapodization of factor 0 in both dimensions. The spectra wereacquired without decoupling of 13C.

For the measurement of the 1,1-ADEQUATE spectrumfor rifamycin S, a refocused pulse sequence using chirppulses15b was employed. The spectrum was acquired in thephase-sensitive mode with data acquisition of 128 scans and256 increments in F1. The delays were set to a correspondingcoupling constants of 145 Hz (H,C) and 50 Hz (C,C). GARPmodulation of 13C was used for decoupling. The spectrumwas processed with 2048 ð 256 data points, using a shiftedsquared sine-bell function of factor 2 in both dimensions.

The 1D-ROE experiments were performed using the sin-gle pulsed-field-gradient echo block for achieving selectiveexcitation. The selective 180° pulse was applied with aGaussian shape (truncated to 5%) and a pulse width of20–60 ms at an attenuation level of 55–69 dB. A mixingtime of 600–700 ms was used for the spin lock period withan attenuation level of 23.88 dB. The spectral width was ca6800 Hz and the number of scans 8. The FIDs were multipliedby an exponential weighting function (lb D 1 Hz) prior toFourier transformation.

Supplementary materialSupplementary material including tables with the character-ization of compounds 5cM, 5cm, 1 (CDCl3) and 2 (CDCl3)and 1D and 2D spectra for all the compounds described inthis paper is available on request from the correspondingauthor.

AcknowledgementsWe thank the Servicio de RMN of the University of Oviedo forallocating measuring time on the NMR spectrometers. We also thankRodolfo Iglesias for technical assistance with the spectrometers.Financial support by Asturpharma SA and projects PC-CIS01-22(FICYT) and PR-01-GE-9 (Grupo de Excelencia FICYT) from the

Consejerıa de Educacion y Cultura del Principado de Asturias isgratefully acknowledged.

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4. (a) Colleter J-C, Gadret M, Goursolle M, Leger J-M. C. R. Acad.Sci. Paris, Ser. C 1974; 279: 1115; (b) Cellai L, Cerrini S, Segre A,Brufani M, Fedeli W, Vaciago A. J. Org. Chem. 1982; 47: 2652,and references cited therein; (c) Bartolucci C, Cellai L, Cerrini S,Lamba D, Segre AL, Brizzi V, Brufani M. Helv. Chim. Acta 1990;73: 185.

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Documents

NMR spectroscopic analysis of new spiro-piperidylrifamycins