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Analysis of Solid-State Carbon-13 NMR Spectra of Polymorphs (Benoxaprofen and Nabilone) and Pseudopolymorphls (Cefazolin)
Analysis of Solid-State Carbon-13 NMR Spectra of Polymorphs (Benoxaprofen and Nabilone) and Pseudopolymorphls (Cefazolin) STEPHEN
R. BYRN*',GEORGEGRAY*,RALPHR.
PFEIFFER5, AND
JAMESFRYEn
Received Februa 21,1984, from the * Department of Medicinal Chemistry and Pharmacognosy Purdue Universit West Lafayelte, IN 47906, Warian Associates, Palo %to, CA 94303, §LillyResearch Laboratories, Indianapolis, IN 46280, and the 'Department of Ckmistry, Colorado State University, Accepted for publication January 9, 1985. Fort Collins, CO 80523.
Abstract 0 The solid-state 13C-.NMR spectra of the polymorphs of
benoxaprofen, nabilone, and cefazolin are reported using the crosspolarization/magic-angle spinning (CP/MAS)technique. In general, the spectra of the diffrent crystal form are different. In favorable cases the spectra of the drug in a pharmaceutical granulation can be discerned. These results provide a preliminary indication that solid-state NMR spectroscopy is a useful techniqlJe for the investigation of drug polymorphs and druqs in their dosage forms. Solid-state NMR spectroscopy has found wide application in the analysis of organic compounds and polymers.'-4 In the area of organic chemistry, solid-state NMR spectroscopy has been used to study the conformation of crystalline organic compounds and to compare the solid-state and solution conformation of m01ecules.~-'~ In addition, solid-state NMR spectroscopy has been used to study biomolecules including uracil, imidazole, and m ~ r p h i n e . ' ~ -This ' ~ study is significant in that it reports on the application of solid-state NMR spectroscopy to the study of drug polymorphs and pseudopolymorphs. The results of this investigation show that the solid-state NMR spectra of polymorphs and pseudopolymorphls are different and indicate that solid-state NMR spectroscopy is a useful method for the study of polymorphs and pseudopolymorphs.
Experimental Section The drugs for this study were obtained from Eli Lilly and Co. and were crystallized as described in the literature. Benoxaprofen gave two polymorphs as shown by powder diffraction, one form showed major powder diffraction peaks at 20 values of 16.3, 23.5, and 25.0 and weak9 peaks a t 18.9 and 20.8 and belongs to space group P2' based on analysis of single crystals. The second form had its major powder diffraction peaks at 20 values of 13.9, 15.8, 17.6, 20.0, 21.5, 24.3, 25.2, and 27.55 and belongs to space group P2Jc based on analysis of single crystals. The granulation of benoxaprofen contains 60% of the drug along with several excipients including starch and polyvinyl pyrrolidone. Nabilone and cefazolin were crystallized as described in the literat~re.'~-'~ The purity of the nabilone polymorphs was checked by powder diffraction. The solid-state NMR spectra of the different forms were obtained using the cross-polarization magic-angle spinning (CP/MAS) technique. Chemical shifts are reported relative to Me,Si via hexamethylbenzene as a secondary reference. The solid-state spectra of benoxaprofen and nabilone WEre measured at Colorado State University on a Nicolet NTC-150 instrument and those of cefazolin were measured at Vaiian on a Varian XL-200 instrument. The spectra were measured without optimization of contact times. The completely decoupled solution 13C NMR spectra were measured for comparison. 0022-3549/85/050@0565$01.OO/O 0 7 985, American Pharmaceutical,4ssociation
Results and Discussion The solid-state NMR spectra of these different crystal forms are shown in Figs. 1-4 and Tables I and 11. The peak heights of the resonances in these spectra are determined by the rate of cross polarization TcH,which varies. Thus, the relative peak heights do not represent the number of carbon atoms contributing to the resonance. Figure 1 shows the solution and solid-state 13C NMR spectra of benoxaprofen. The solid-state spectra of crystal forms I and I1 are consistent with the solution spectrum and show Carbon-13 resonances which have approximately the same chemical shift. However, the largest peak in the spectrum of form I appears to be shifted slightly in form 11. This may be attributed to slight shifts in the positions of the resonances due to the 13 aromatic carbon atoms contributing to this signal, resulting in a change in appearance of the peak. Table I lists the chemical shifts of the CH,, CH, and COOH carbon atoms (carbon atoms 3, 2, and 1). It is clear from this table that the chemical shifts of carbon atoms 3 and 2 change on going from form I to form 11. The spectrum of a mixture of forms I and I1 is completely consistent with this argument. In addition, the shoulder and double peak for the CH and CH, carbon atoms indicate that solid-state NMR can be used as a qualitative method to determine whether a mixture of the two forms is present. However, this spectrum of the mixture indicates that, with these instrumental conditions, the different carbon atoms do not give identical responses, and thus the intensity-concentration curve is not linear. These results are consistent with reports in the literature which show: ( a ) that small changes in the chemical shifts can be attributed to intermolecular shielding terms while larger shifts can be attributed to changes in conformation,8.''1 l 4 and ( b ) that quantitative analysis is in general not possible with spectra being measured using usual CP/MAS techniques without careful calibration of each peak using internal standards or without optimization of the contact times.' The spectrum of the granulation of benoxaprofen is distinct (see Fig. 1)and shows three peaks in the range of 50-100 ppm which are attributable to the excipients. Other than these signals the spectrum is virtually identical to that of form 11. Table I shows that the chemical shifts of the CH and CH, carbon atoms are virtually identical to form 11. This data indicates that benoxaprofen in the granulation is in form I1 and is consistent with results from X-ray powder diffraction. Figures 2 and 3 show the solid-state spectra of two polymorphs of nabilone. The sharpness of the signals argues that these spectra are of crystalline r n a t e r i a l ~ $ . ~however, .~~ the presence of an amorphous material cannot be ruled out. Figure 3 shows an expansion of the 0 to 50 ppm region of the spectra shown in Fig. 2. Table I1 lists the chemical shifts of the peaks from these two polymorphs. Comparison of the solid-state Journal of Pharmaceutical Sciences / 565 Vol. 74, No. 5, May 1985
a
Figure 2-Solution and solid state I3C-NMR spectra of nabilone. Key: (a) solution spectrum; (b) solid-state spectrum of nabilone I; (c) solidstate spectrum of nabilone 11.
answered by examining Table 11. There are nine peaks in the nabilone I spectrum which do not correspond to any peaks in the spectrum of nabilone 11, and there are five peaks unique to the nabilone I1 spectrum. These data indicate that each of the 0
Figure 1-Solution and solid-state 13C-NMRspectra of benoxaprofen. Key: (a) solution spectrum; (b) solid-state spectrum of form I; (c) solidstate spectrum of form 11; (d) solid-state spectrum of a 1:l mixture of forms l and I/; (e) solid-state spectrum of a granulation containing 66% benoxaprofen.
spectrum of nabilone I with nabilone I1 shows that solid-state NMR can be used to qualitatively determine which polymorphs are present in a binary mixture (i.e., I with 11).
For complicated spectra such as these the question arises as to whether the individual polymorphs are pure (i.e., is form 11 actually a mixture of forms I and 11). This question can be 566 /Journal of Pharmaceutical Sciences Vol. 74, No. 5, May 1985
Figure 3-Expanded (b) nabilone I/.
solid-state spectra of nabilone. Key: (a) nabilone /;
Table I-Chemical Shifts (ppm) of the Carbon Atoms in the Various Polymorphs of Benoxaprofen in the Solid State
a
Carbon Atom
Form I
Form II
Granulation
CH3 (carbon-3) CH (carbon-2)
19.7 46.3 128.5 182.6
15.5 42.5 126.8 182.3
15.6 42.7 127.0 182.6
Maximum peak COOH (carbon-1)
Table 11-Chemicel Shifts of the Carbon Atoms in the Various Polymorphsof Nabilone in the Solid State
Nabilone I
Nabilone II
17.455 20.8 24.5 26.5 27.9 30.0 31.8 35.2 36.2 38.1 42.2 44.4 45.8 78.0 106.4 157.3 218.3
15.0 19.8 24.0 25.3 25.9 28.1 29.4 30.8 31.8 32.2 35.9 38.1 43.4 46.2 77.5 105.7 106.7 157.2 217.6
200
150
10Cl
50
0 PPm
Figure 4-Solution and solid-state '3C-NMR spectra of cefazolin. Key: (a) peak assignments from solution spectrum; (b) solid-state spectrum of the pentahydrate; (c) solid-state spectrum of the sesquihydrate; (d) solidstate spectrum of the monohydrate; (e) solid-state spectrum of the amorphous form.
polymorphs is pure, which is consistent with X-ray powder diffraction results. The spectra of the various hydrated crystal forms of cefazolin (which are pseudopolymorphs) along with the solution spectrum are shown in Fig. 4. X-ray powder diffraction shows that these forms have different crystal structures. The assignments of the carbon resonances in the solution spectrum (Fig. 4a) are taken from the literature?' Disitinct signals can be seen for all carbon atoms in solution and in the solid-state spectrum of the pentahydrate, although the positions of the peaks are slightly shifted. The spectrum of the sesquihydrate does not show distinct signals for all of the aromatic carbon atoms. There are
only two broad peaks in the 180-200 ppm range. In addition, the positions of these two resonances are different from the peak positions in the pentahydrate. The spectrum of the monohydrate is slightly different from the sesquihydrate and the peak positions are again different. The amorphous form shows broadened peaks, as expected based on reports in the literat ~ r e ? , These ~ , ~ ~spectra indicate that solid-state NMR spectroscopy can possibly be used to qualitatively determine which hydrated form is present. In addition, as with nabilone, these data indicate that one could possibly use solid-state NMR to assay semiquantitatively binary mixtures of the various hydrates. The data on benoxaprofen, nabilone, and cefazolin allowed the following conclusions to be made. First, different crystal forms of drugs have different solid-state NMR spectra. Second, solid-state NMR spectroscopy can be used to determine whether mixtures of two forms are present and to identify the forms involved in these mixtures if the spectra of the pure components are available. However, it appears from these data as well as data in the literature that unless proton longitudinal and rotating frame relaxation behavior is investigated, solidstate NMR spectroscopy is not yet useful for quantitation of mixtures. Third, the data on benoxaprofen indicate that, in favorable cases, solid-state NMR spectroscopy can be used to study dosage forms and to determine the crystalline forms present in these dosage forms. Fourth, the data on cefazolin suggest the tentative conclusion that amorphous materials will show broad peaks and can, in favorable cases, be discriminated from crystalline forms. Future prospects for solid-state NMR spectroscopy include the study of the interactions of drugs with polymer matrices in dosage forms, I5N NMR spectroscopy, and the quantitation of mixtures of physical forms.
References and Notes 1. Hays, G. R.; Huis, R.; Coleman, B.; Clague, D.; Verhoeven, J. W.; Rob, F. J. Am. Chem. SOC.1981,103, 5140-5146. 2. Alewany, L. B.; Grant, D. M.; Pugmire, R. J.; Alger, T. D.; Zilm, K. W. J . Am. Chem. SOC.1983,105,2133-2141 and 2142-2147.
Journal of Pharmaceutical Sciences / 567 Vol. 74, No. 5, May 1985
3. Yannoni, C. S.Acc. Chem. Res. 1982,15,201-208. 4. Lyerla, J. R.; Yannoni, C. S.; Fyfe, C. A. Acc. Chem. Res. 1982, 15,208-216. 5. VanderHart, D. L.; Earl, W. L.; Garroway, A. N. J. Mag. Res. 1981,44,361-401. 6 . VanderHart, D. L. J. Mag. Res. 1981,44,117-125. 7. Ripmeester, J. A. Chem. Phys. Lett. 1980, 74,536-538. McKay, R. A.; Stults, B.R.; Schaefer, 8. Steger, T. R.; Stejskal, E. 0.; J. Tetrahedron Left. 1979,295-296. 9. Hays, G. R. Analyst (London) 1982,107,241-252. 10. Kessler, H.; Zimmermann, G.; Forster, H.; Engle, J.; Oepen, G.; Sheldrick, W. S.; Angew. Chem. Znt. Ed. Engl. 1981, 20, 10531055. 11. Hill, H. D. W.; Zeus, A. P.; Jacobus, J. J. Am. C k m . Soc. 1979, 101,7090-7091. 12. Shiau, W.I.; Duesler, E. N.; Paul, I. C.; Curtin, D. Y.; Blann, W. G.; Fyfe, C. A. J. Am. Chem. SOC.1980,102,4546-4548. 13. McDowell, C. A.; Naito, A.; Scheffer, J. R.; Wong, Y. F. Tetrahedron Lett. 1981,4779-4782. 14. Dalling, D. K.; Zilm, K. W.; Grant, D. M.; Heeschen, W. A.; Horton, W. J.; Pugmire, R. J. J. Am. Chem. SOC.1981,103,4817-4824. 15. Ganapathy, S.;Naito, A.; McDowell, C. A.; J. Am. Chem. SOC. 19819103,6011-6015.
568 /Journal of Pharmaceutical Sciences Vof. 74, No. 5, May 1985
16. Haberkorn, R. A.; Stark, R. E.; VanWilligen, H.; Griffin, R. G. J. Am. Chem. SOC.1981,103,2534-2539. 17. Habison, G.; Herzfeld, J.; Griffin, R. G. J. Am. Chem. SOC.1981, 103,4752-4754. 18. Gall, C. M.; DiVerdi, J. A.; Opella, S. J. J. Am. Chem. Soc. 1981, 103,5039-5043. 19. Thakkar, A. L.; Hirsch, C. A.; Page, J. G. J. Pharrn. Pharmacol. 1977,29,783-784. 20. Archer, R. A.; Blanchard, W. B.; Day, W. A.; Johnson, D. W.; Lavagnino, E. R.; Ryan, C. W.; Baldwin, J. E. J. Org. Chem. 1977, 42,2277-2284. 21. Kariyone, K.;Harada, H.; Kurita, M.; Takano, T. J. Antibiot. 1970,23, 131-136. 22. Tori, K.; Nishikawa, J.; Takeuchi, Y. Tetrahedron Left. 1981, 2793-2796. 23. Kolodziejski, W.; Frye, J. S.; Maciel, G. E. Anal Chem. 1982,54, 1419-1424.
Acknowledgments This research was supported by Eli Lilly and Co. and we are grateful to the Colorado State University Regional NMR Center, funded by National Science Foundation Grant No. CHE 78-18581.
R. BYRN*',GEORGEGRAY*,RALPHR.
PFEIFFER5, AND
JAMESFRYEn
Received Februa 21,1984, from the * Department of Medicinal Chemistry and Pharmacognosy Purdue Universit West Lafayelte, IN 47906, Warian Associates, Palo %to, CA 94303, §LillyResearch Laboratories, Indianapolis, IN 46280, and the 'Department of Ckmistry, Colorado State University, Accepted for publication January 9, 1985. Fort Collins, CO 80523.
Abstract 0 The solid-state 13C-.NMR spectra of the polymorphs of
benoxaprofen, nabilone, and cefazolin are reported using the crosspolarization/magic-angle spinning (CP/MAS)technique. In general, the spectra of the diffrent crystal form are different. In favorable cases the spectra of the drug in a pharmaceutical granulation can be discerned. These results provide a preliminary indication that solid-state NMR spectroscopy is a useful techniqlJe for the investigation of drug polymorphs and druqs in their dosage forms. Solid-state NMR spectroscopy has found wide application in the analysis of organic compounds and polymers.'-4 In the area of organic chemistry, solid-state NMR spectroscopy has been used to study the conformation of crystalline organic compounds and to compare the solid-state and solution conformation of m01ecules.~-'~ In addition, solid-state NMR spectroscopy has been used to study biomolecules including uracil, imidazole, and m ~ r p h i n e . ' ~ -This ' ~ study is significant in that it reports on the application of solid-state NMR spectroscopy to the study of drug polymorphs and pseudopolymorphs. The results of this investigation show that the solid-state NMR spectra of polymorphs and pseudopolymorphls are different and indicate that solid-state NMR spectroscopy is a useful method for the study of polymorphs and pseudopolymorphs.
Experimental Section The drugs for this study were obtained from Eli Lilly and Co. and were crystallized as described in the literature. Benoxaprofen gave two polymorphs as shown by powder diffraction, one form showed major powder diffraction peaks at 20 values of 16.3, 23.5, and 25.0 and weak9 peaks a t 18.9 and 20.8 and belongs to space group P2' based on analysis of single crystals. The second form had its major powder diffraction peaks at 20 values of 13.9, 15.8, 17.6, 20.0, 21.5, 24.3, 25.2, and 27.55 and belongs to space group P2Jc based on analysis of single crystals. The granulation of benoxaprofen contains 60% of the drug along with several excipients including starch and polyvinyl pyrrolidone. Nabilone and cefazolin were crystallized as described in the literat~re.'~-'~ The purity of the nabilone polymorphs was checked by powder diffraction. The solid-state NMR spectra of the different forms were obtained using the cross-polarization magic-angle spinning (CP/MAS) technique. Chemical shifts are reported relative to Me,Si via hexamethylbenzene as a secondary reference. The solid-state spectra of benoxaprofen and nabilone WEre measured at Colorado State University on a Nicolet NTC-150 instrument and those of cefazolin were measured at Vaiian on a Varian XL-200 instrument. The spectra were measured without optimization of contact times. The completely decoupled solution 13C NMR spectra were measured for comparison. 0022-3549/85/050@0565$01.OO/O 0 7 985, American Pharmaceutical,4ssociation
Results and Discussion The solid-state NMR spectra of these different crystal forms are shown in Figs. 1-4 and Tables I and 11. The peak heights of the resonances in these spectra are determined by the rate of cross polarization TcH,which varies. Thus, the relative peak heights do not represent the number of carbon atoms contributing to the resonance. Figure 1 shows the solution and solid-state 13C NMR spectra of benoxaprofen. The solid-state spectra of crystal forms I and I1 are consistent with the solution spectrum and show Carbon-13 resonances which have approximately the same chemical shift. However, the largest peak in the spectrum of form I appears to be shifted slightly in form 11. This may be attributed to slight shifts in the positions of the resonances due to the 13 aromatic carbon atoms contributing to this signal, resulting in a change in appearance of the peak. Table I lists the chemical shifts of the CH,, CH, and COOH carbon atoms (carbon atoms 3, 2, and 1). It is clear from this table that the chemical shifts of carbon atoms 3 and 2 change on going from form I to form 11. The spectrum of a mixture of forms I and I1 is completely consistent with this argument. In addition, the shoulder and double peak for the CH and CH, carbon atoms indicate that solid-state NMR can be used as a qualitative method to determine whether a mixture of the two forms is present. However, this spectrum of the mixture indicates that, with these instrumental conditions, the different carbon atoms do not give identical responses, and thus the intensity-concentration curve is not linear. These results are consistent with reports in the literature which show: ( a ) that small changes in the chemical shifts can be attributed to intermolecular shielding terms while larger shifts can be attributed to changes in conformation,8.''1 l 4 and ( b ) that quantitative analysis is in general not possible with spectra being measured using usual CP/MAS techniques without careful calibration of each peak using internal standards or without optimization of the contact times.' The spectrum of the granulation of benoxaprofen is distinct (see Fig. 1)and shows three peaks in the range of 50-100 ppm which are attributable to the excipients. Other than these signals the spectrum is virtually identical to that of form 11. Table I shows that the chemical shifts of the CH and CH, carbon atoms are virtually identical to form 11. This data indicates that benoxaprofen in the granulation is in form I1 and is consistent with results from X-ray powder diffraction. Figures 2 and 3 show the solid-state spectra of two polymorphs of nabilone. The sharpness of the signals argues that these spectra are of crystalline r n a t e r i a l ~ $ . ~however, .~~ the presence of an amorphous material cannot be ruled out. Figure 3 shows an expansion of the 0 to 50 ppm region of the spectra shown in Fig. 2. Table I1 lists the chemical shifts of the peaks from these two polymorphs. Comparison of the solid-state Journal of Pharmaceutical Sciences / 565 Vol. 74, No. 5, May 1985
a
Figure 2-Solution and solid state I3C-NMR spectra of nabilone. Key: (a) solution spectrum; (b) solid-state spectrum of nabilone I; (c) solidstate spectrum of nabilone 11.
answered by examining Table 11. There are nine peaks in the nabilone I spectrum which do not correspond to any peaks in the spectrum of nabilone 11, and there are five peaks unique to the nabilone I1 spectrum. These data indicate that each of the 0
Figure 1-Solution and solid-state 13C-NMRspectra of benoxaprofen. Key: (a) solution spectrum; (b) solid-state spectrum of form I; (c) solidstate spectrum of form 11; (d) solid-state spectrum of a 1:l mixture of forms l and I/; (e) solid-state spectrum of a granulation containing 66% benoxaprofen.
spectrum of nabilone I with nabilone I1 shows that solid-state NMR can be used to qualitatively determine which polymorphs are present in a binary mixture (i.e., I with 11).
For complicated spectra such as these the question arises as to whether the individual polymorphs are pure (i.e., is form 11 actually a mixture of forms I and 11). This question can be 566 /Journal of Pharmaceutical Sciences Vol. 74, No. 5, May 1985
Figure 3-Expanded (b) nabilone I/.
solid-state spectra of nabilone. Key: (a) nabilone /;
Table I-Chemical Shifts (ppm) of the Carbon Atoms in the Various Polymorphs of Benoxaprofen in the Solid State
a
Carbon Atom
Form I
Form II
Granulation
CH3 (carbon-3) CH (carbon-2)
19.7 46.3 128.5 182.6
15.5 42.5 126.8 182.3
15.6 42.7 127.0 182.6
Maximum peak COOH (carbon-1)
Table 11-Chemicel Shifts of the Carbon Atoms in the Various Polymorphsof Nabilone in the Solid State
Nabilone I
Nabilone II
17.455 20.8 24.5 26.5 27.9 30.0 31.8 35.2 36.2 38.1 42.2 44.4 45.8 78.0 106.4 157.3 218.3
15.0 19.8 24.0 25.3 25.9 28.1 29.4 30.8 31.8 32.2 35.9 38.1 43.4 46.2 77.5 105.7 106.7 157.2 217.6
200
150
10Cl
50
0 PPm
Figure 4-Solution and solid-state '3C-NMR spectra of cefazolin. Key: (a) peak assignments from solution spectrum; (b) solid-state spectrum of the pentahydrate; (c) solid-state spectrum of the sesquihydrate; (d) solidstate spectrum of the monohydrate; (e) solid-state spectrum of the amorphous form.
polymorphs is pure, which is consistent with X-ray powder diffraction results. The spectra of the various hydrated crystal forms of cefazolin (which are pseudopolymorphs) along with the solution spectrum are shown in Fig. 4. X-ray powder diffraction shows that these forms have different crystal structures. The assignments of the carbon resonances in the solution spectrum (Fig. 4a) are taken from the literature?' Disitinct signals can be seen for all carbon atoms in solution and in the solid-state spectrum of the pentahydrate, although the positions of the peaks are slightly shifted. The spectrum of the sesquihydrate does not show distinct signals for all of the aromatic carbon atoms. There are
only two broad peaks in the 180-200 ppm range. In addition, the positions of these two resonances are different from the peak positions in the pentahydrate. The spectrum of the monohydrate is slightly different from the sesquihydrate and the peak positions are again different. The amorphous form shows broadened peaks, as expected based on reports in the literat ~ r e ? , These ~ , ~ ~spectra indicate that solid-state NMR spectroscopy can possibly be used to qualitatively determine which hydrated form is present. In addition, as with nabilone, these data indicate that one could possibly use solid-state NMR to assay semiquantitatively binary mixtures of the various hydrates. The data on benoxaprofen, nabilone, and cefazolin allowed the following conclusions to be made. First, different crystal forms of drugs have different solid-state NMR spectra. Second, solid-state NMR spectroscopy can be used to determine whether mixtures of two forms are present and to identify the forms involved in these mixtures if the spectra of the pure components are available. However, it appears from these data as well as data in the literature that unless proton longitudinal and rotating frame relaxation behavior is investigated, solidstate NMR spectroscopy is not yet useful for quantitation of mixtures. Third, the data on benoxaprofen indicate that, in favorable cases, solid-state NMR spectroscopy can be used to study dosage forms and to determine the crystalline forms present in these dosage forms. Fourth, the data on cefazolin suggest the tentative conclusion that amorphous materials will show broad peaks and can, in favorable cases, be discriminated from crystalline forms. Future prospects for solid-state NMR spectroscopy include the study of the interactions of drugs with polymer matrices in dosage forms, I5N NMR spectroscopy, and the quantitation of mixtures of physical forms.
References and Notes 1. Hays, G. R.; Huis, R.; Coleman, B.; Clague, D.; Verhoeven, J. W.; Rob, F. J. Am. Chem. SOC.1981,103, 5140-5146. 2. Alewany, L. B.; Grant, D. M.; Pugmire, R. J.; Alger, T. D.; Zilm, K. W. J . Am. Chem. SOC.1983,105,2133-2141 and 2142-2147.
Journal of Pharmaceutical Sciences / 567 Vol. 74, No. 5, May 1985
3. Yannoni, C. S.Acc. Chem. Res. 1982,15,201-208. 4. Lyerla, J. R.; Yannoni, C. S.; Fyfe, C. A. Acc. Chem. Res. 1982, 15,208-216. 5. VanderHart, D. L.; Earl, W. L.; Garroway, A. N. J. Mag. Res. 1981,44,361-401. 6 . VanderHart, D. L. J. Mag. Res. 1981,44,117-125. 7. Ripmeester, J. A. Chem. Phys. Lett. 1980, 74,536-538. McKay, R. A.; Stults, B.R.; Schaefer, 8. Steger, T. R.; Stejskal, E. 0.; J. Tetrahedron Left. 1979,295-296. 9. Hays, G. R. Analyst (London) 1982,107,241-252. 10. Kessler, H.; Zimmermann, G.; Forster, H.; Engle, J.; Oepen, G.; Sheldrick, W. S.; Angew. Chem. Znt. Ed. Engl. 1981, 20, 10531055. 11. Hill, H. D. W.; Zeus, A. P.; Jacobus, J. J. Am. C k m . Soc. 1979, 101,7090-7091. 12. Shiau, W.I.; Duesler, E. N.; Paul, I. C.; Curtin, D. Y.; Blann, W. G.; Fyfe, C. A. J. Am. Chem. SOC.1980,102,4546-4548. 13. McDowell, C. A.; Naito, A.; Scheffer, J. R.; Wong, Y. F. Tetrahedron Lett. 1981,4779-4782. 14. Dalling, D. K.; Zilm, K. W.; Grant, D. M.; Heeschen, W. A.; Horton, W. J.; Pugmire, R. J. J. Am. Chem. SOC.1981,103,4817-4824. 15. Ganapathy, S.;Naito, A.; McDowell, C. A.; J. Am. Chem. SOC. 19819103,6011-6015.
568 /Journal of Pharmaceutical Sciences Vof. 74, No. 5, May 1985
16. Haberkorn, R. A.; Stark, R. E.; VanWilligen, H.; Griffin, R. G. J. Am. Chem. SOC.1981,103,2534-2539. 17. Habison, G.; Herzfeld, J.; Griffin, R. G. J. Am. Chem. SOC.1981, 103,4752-4754. 18. Gall, C. M.; DiVerdi, J. A.; Opella, S. J. J. Am. Chem. Soc. 1981, 103,5039-5043. 19. Thakkar, A. L.; Hirsch, C. A.; Page, J. G. J. Pharrn. Pharmacol. 1977,29,783-784. 20. Archer, R. A.; Blanchard, W. B.; Day, W. A.; Johnson, D. W.; Lavagnino, E. R.; Ryan, C. W.; Baldwin, J. E. J. Org. Chem. 1977, 42,2277-2284. 21. Kariyone, K.;Harada, H.; Kurita, M.; Takano, T. J. Antibiot. 1970,23, 131-136. 22. Tori, K.; Nishikawa, J.; Takeuchi, Y. Tetrahedron Left. 1981, 2793-2796. 23. Kolodziejski, W.; Frye, J. S.; Maciel, G. E. Anal Chem. 1982,54, 1419-1424.
Acknowledgments This research was supported by Eli Lilly and Co. and we are grateful to the Colorado State University Regional NMR Center, funded by National Science Foundation Grant No. CHE 78-18581.