Application of LC-NMR for the quick identification of process. related impurities in the synthesis of Docetaxel intermediate

Transcription

1 124 Application of LC-NMR for the quick identification of process related impurities in the synthesis of Docetaxel intermediate 5.1 Introduction Taxol, a naturally occurring diterpene, has overwhelming evidence of the chemotherapeutic value clinical trials against cancer by the year showed [1 & 2] in the Consequently, a variety of synthetic analogues of taxol were synthesized and examined for their ability to inhibit microtubules disassembly and their potencies were compared good correlation [3[. between disassembly assay Some of these compounds showed a cytotoxicity and the microtubule [4]. In taxoid family Paclitaxel (i.e. Taxol) which was initially named as Taxol and Docetaxel (i.e. Taxotere) (Scheme 5.1) are currently considered to be two of the most exciting drugs in the cancer chemotherapy. activity These two molecules exhibit significant antitumor [5]. Paclitaxel (i.e. Taxol) is used for the treatment of breast, ovarian, lung, bladder, prostate, melanoma, esophageal, as well as other types of solid tumor cancers. Taxotere can be used for lonely or with combination mode for the following cancers Breast Cancer, Advanced Non-Small Cell Lung Cancer, Metastatic Androgen-Independent Prostate Cancer Advanced Gastric/GE Junction,Locally Advanced Head and Neck Cancer

2 125 Scheme Structures of Paclitaxel and Docetaxel Present Work: A number of taxol analogs have been described by F. Guueritte- Voegelein et al [6] Docetaxel (DCT) is an antineoplastic agent. The Docetaxel was synthesized and marketed by Dr. Reddy s Laboratories Ltd, for the treatment of cancer. The total synthesis of Docetaxel, is very lengthy and difficult to manufacture. There are several reports available for the synthesis Docetaxol from naturally occuring 10deacetylbaccatin III (10-DAB III), which is readily available from the renewable leaves of Taxus baccata (European Yew) and it was described in Scheme 5.2 [7].

3 126 Scheme 5.2 Synthetic scheme of Docetaxel For the preparation of Docetaxol, the 10-DAB III hydroxyl groups at 7 and 10 positons have to be protected. Due to the steric hinderence the hydroxyl group at 1 position will not react with the protecting groups. However the present reaction conditions are not favorable for the substitution at 13 position. Based on the literature precedence N, N-carbodiimidazole was chosen as protecting group. During the process development / optimization of Docetaxel, few byproducts were observed in the preparation of DCT-1. The synthesis of DCT-1 was given in Scheme 5.3. These by-products were observed at higher percentages when acetone was used as a solvent. To know the structure of these by-products LC-MS and LC-NMR studies were used without isolation. The characterization of these by-products in the synthesis of docetaxel process development is presented in this chapter.

4 127 Scheme Preparation of DCT-1 Experimental Samples Crude sample of DCT-1 was subjected for the identification of the by-products. Samples of pure 10-DAB III and DCT-1 were used to generate spectral data for the comparison with those of the byproducts. Pure samples of 10-DAB III and DCT-1 were obtained from Dr. Reddy s Laboratories Ltd., Hyderabad, India Analytical HPLC An in-house HPLC method was developed for the separation of the impurities and the products. The HPLC conditions are shown in Table 5.1.

5 128 Table 5.1 Column Solvent A Analytical HPLC conditions 250mm 4.6mm, 5 μm, RPB (Highchrom, manufactured by Hichrom Ltd., Berkshire, UK) Water (HPLC grade) Solvent B Acetonitrile (HPLC grade) Gradient Program 0/35, 15/65, 25/75, 30/95, 35/100, 39/100 and (T / %B) 40/35 for post run Flow rate 1.0 ml / min Detection 230 nm Injection volume 10 μl Diluent 1:1 (water : CH3CN) Mass spectrometry A HPLC method suitable for LC-MS was developed by using Ammonium Acetate buffer. The LC conditions are shown in Table 5.2. Table 5.2 LC-MS conditions Column Inertsil DS 4.6mm 250 mm, 5.0 m Solvent A 0.01M Ammonium Acetate (ph 3.2 with Acetic Acid) Solvent B Acetonitrile (HPLC grade) Isocratic 50:50 Flow rate 1.0 ml / min Detection 230 nm Injection volume 10 μl Diluents 1 : 1 (water : CH3CN)

6 LC-NMR spectroscopy The regular HPLC method (cf. Table 5.1) was modified for the LCNMR analysis to reduce the retention time. The LC conditions are shown in Table 5.3. Table 5.3 Column LC conditions for LC-NMR Solvent B Hypersil BDS C18, 205mm 4.6 mm, 5.0 m (Thermo Hypersil-Keystone, Germany) 0.01M Potassium dihydrogen phosphate (ph 3.2 with H3P=4) in D2 Acetonitrile (LC-NMR grade) Isocratic 50:50 Flow rate 1.0 ml / min Detection 230 nm Solvent A Injection volume 10 μl Diluents 1 : 1 (water : CH3CN) NMR Spectroscopy : The LC - NMR spectra were recorded in at 300K using Varian Unity INVA 500 MHz NMR spectrometer, equipped with a Varian 3mm Interchangeable Flow Cell (IFC) 19F{13C/15N} cell. - pulse field gradient (PFG) probe fitted with a 60 l flow The chemical shift values were reported on the scale in ppm, relative to acetonitrile ( = 1.95). Standard lc1d pulse sequence provided by Varian was used, 500 MHz -19F{13C/15N} 60 l PFG microflow Probe. The LC NMR was run by using stop flow method.

7 NMR spectroscopy The NMR experiments, for the pure 10-DAB III and DCT-1, such as, 13C, gdqcsy, ghsqc, ghmbc and NESY were performed on Mercury plus 400 MHz and Unity Inova 500 MHz, Varian instruments, at 25 C in DMS-d Results and discussions Detection and Separation of by-products. The analytical HPLC chromatogram of the crude DCT-1 was obtained by using the HPLC method (cf. Section 5.2.2) is shown in Figure 5.1. Two unknown by-products were observed at 7.81 and 9.35 min. Henceforth, these by-products will be referred as BP-I and BP-II. The starting compounds, (10-DAB III) and the product (DCT-1) eluted at 5.68 and min respectively. Figure 5.1 Analytical HPLC chromatogram of crude DCT-1

8 131 Figure 5.2 LC-MS chromatogram of crude DCT-1 (a) TIC spectrum (b) UV Chromatogram The LC-MS data was generated (Figure 5.2) to obtain the molecular ion information of BP-I and BP-II. Interestingly, both BP-I and BP-II displayed the same molecular ion, at m/z 639 (Figures 5.3 and 5.4) [M+H]+, corresponding to mono protected 10-DAB III. While the product DCT-1 has both 7 and 10-hydroxyl groups are protected. The proposed structures of BP-I and BP-II are shown in Scheme 5.4. LC-NMR technique is employed for the unambiguous identification of by-products, BP-I and BP-II.

9 132 Scheme Proposed structures of by-products Structural chemistry of 10-DAB II, DCT-1, BP-I and BP-II As a first step, the NMR structural investigation of DCT-1 is taken up. This would facilitate easier identificaion of the structures of by-products. The positive ES-MS spectrum (Figure 5.5) of DCT-1 gave molecular ion at 733.3, confirms substitution of two imidazolyl moieties on 10-DAB III. The proton NMR assignments of 10-DAB III were already reported [8]. The, 13C, DQCSY and HMBC NMR data of DCT-1 generated in DMS-d6. The spectra are shown in Figures 5.6 to 5.9. The and 13C NMR data of 10-DAB III, was also generated in DMS-d6 for comparison with DCT-1 (Figures 5.10 and 5.11).

10 133 Figure 5.3 Positive ES-MS spectrum of 5.36min Figure 5.4 Positive ES-MS spectrum of 6.69min

11 134 Figure 5.5 Figure 5.6 Positive ES-MS spectrum of DCT-1 NMR spectrum of DCT-1 in DMS-d6.

12 135 Figure C NMR spectrum of DCT-1 in DMS-d6. Figure 5.8 CSY spectrum of DCT-1 in DMS-d6.

13 136 Figure 5.9 HMBC spectrum of DCT-1 in DMS-d6. The numbering scheme for 10-DAB III and DCT-1 used in the discussion is shown in Scheme 5.5. Scheme 5.5 Structures of 10-DAB III and DCT-1 with numbering The NMR assignments (Table 5.4) of 10-DAB III and DCT-1 were compared. The substitution of imidazolyl groups, at C-7 and C-10, lead to changes in the chemical shifts of protons and carbons in DCT1. The chemical shifts of methine proton (carbon) at C-7 position in 10-DAB-III, 4.11 ppm (70.94 ppm) have shifted to 5.62 ppm (75.85

14 137 ppm) in DCT-1. Similarly, the proton (carbon) chemical shifts at C-10 positions in 10-DAB-III, 5.13 ppm (74.39 ppm), have shifted to 6.39 ppm (78.72 ppm) in DCT-1. These chemical shift perturbations due to substitution can be clearly seen in the comparison of and spectra (Figures 5.10 and 5.11). 13C NMR It is clear that these changes in chemical shifts of the protons (and carbon) at C7 and C-10 can be used as a diagnostic tool for the identification of the site of the imidazolyl substitution. Figure 5.10 NMR spectra overlay of 10-DAB III & DCT-1

15 Figure 5.11 (bottom) Table C NMR spectra overlay 10-DAB III (top) & DCT-1 NMR assignments of 10-DAB III and DCT-1 10-DAB No H (ppm) DCT-1 13C (ppm) (ppm) 13C (ppm) Ha Hb H H

16 H 2H H 3H 3H 3H Ha Hb 3H 2H 2H ,29 26, The NMR assignments of 10-DAB III and DCT-1 (both in DMS-d6) and LC-NMR along with the BP-I and BP-II were shown in Table 5.5, between ppm.

17 Table NMR Assignments of 10-DAB III, DCT-1, BP-I and BP-II from LC-NMR 10-DAB III No ,29 26, The DCT-1 BP-I BP-II (ppm) (ppm) (ppm) (ppm) H H overlaid LC-NMR spectra of DCT-1 and 10-DAB-III are shown in Figure The chemical shift changes due to substitution discussed earlier are indicated by arrows in the Figure Having the NMR assignments of 10-DAB III and DCT-1 as basis, the LC- NMR data of arising from LC peaks due to BP-I and BP-II has been analyzed to arrive at their structures. The LC NMR data of BP-I and BP-II was compared with those of the 10-DAB III and DCT-1.

18 141 Figure verlay of LC-NMR spectra of DCT-I with 10-DAB III Structure confirmation of BP-I The NMR spectra of DCT-1, 10-DAB III and BP-I obtained by LC-NMR are compared in Figure A baseline drift is observed in the range of ppm. This is due to the presaturation of the signals due to acetonitrile and water at 1.9 and 4.2 ppm respectively. The aromatic region of BP-I has showed signals at 7.05 (, s), 7.50 (, s) and 8.25 ppm(, s) in addition to the signals due to phenyl moiety (Figure 5.13). This observation showed that there is only one imidazolyl moiety in the BP-I which is in conformity with the molecular weight information from mass spectrometry.

19 142 Figure verlay-lc-nmr spectra of DCT-1, BP-I and 10-DAB III The following significant changes can be observed in the chemical shifts of BP-I. The singlet at 5.20 ppm which is due to methine proton at C-10 in the 10-DAB III spectrum has shifted to 6.50 ppm in BP-I. As discussed earlier, this is diagnostic of substitution of imidazolyl group at C-10. Interestingly, the chemical shift of methine proton at C-10 in DCT-1 is 6.39 ppm which is quite comparable to the chemical shift in BP-I. No other significant changes were observed in the spectrum of BP-I. All these observations confirm the structure of BP-I to be 10-imidazolyl substituted 10-DAB III.

20 143 Scheme Structures of 10-DAB III and BP-I Structure confirmation of BP-II The NMR spectra of DCT-1, 10-DAB-III and BP-II obtained by LC-NMR are compared in Figure Figure 5.14.verlay-LC-NMR spectra of DCT-1, BP-II and 10-DAB III

21 144 The aromatic region of BP-II has showed signals at 7.00 (, s), 7.40 (, s) and 8.15 (, s) in addition to the signals due to phenyl moiety (Figure 5.13). This observation showed that there is only one imidazolyl moiety in BP-II which is in conformity with the molecular weight information from mass spectrometry. The following significant changes can be observed in the chemical shifts of BP-II. The proton NMR spectrum of aliphatic region of BP-II showed a signal at 5.44 ppm (doublet of a doublet) integrating for one proton. This could be assigned due to the methine proton connected to C-7. The corresponding methine proton in 10-DAB III which is expected at ~4.1 ppm could not be seen as it is under the signal due to water, and hence cannot be seen. The C-7 methine proton signal was observed at 4.11pm in NMR spectrum collected in DMS-d6. The remaining protons didn t show significant chemical shift changes. Interestingly, the chemical shift of methine proton at C-7 in DCT-1 is 5.55 which is quite comparable to the chemical shift in BP-II. All these observations confirm the structure of BP-II to be 7imidazolyl substituted 10-DAB III.

22 145 Scheme 5.7 Structure of 10-DAB III and BP-II 5.5 Formation of by-products The synthesis of DCT-1 was to protect 7 and 10 positions of 10- DAB III with N, N-carbodiimidazole. The reaction is shown in Scheme 5.3. The elucidated structures of BP-I and BP-II suggest that these products have formed due to partial protection at either 7 or 10 in 10DAB III (Scheme 5.8) N H H3C CH3 H CH3 H H N, N-Carbonyl dimida zole E thyl Aceta te RT 3.5hr H N N H 3C H + H H H H3C 10 DA B H H H3 C H3 C N N H3C CH3 N N H + H H3C DCT-1 Scheme 5.8 N Formation of by-products BP-I and BP-II H3 C

23 Conclusion The LC-MS data has helped in the identification of the tentative structures of the by-products formed during the reaction. The LC- NMR has helped in the quick identification of the structures of these by-products unambiguously without resorting to isolation of the by Preparative HPLC.

24 REFERENCES 1 Rowinsky, E.K., Cazenave, L., and Donehower, R., J. Natl. Cancer Inst., 82, 1990, Guéritte - Voegelein,F., Guénard, D., Lavelle, F., Le Goff, M., Mangatal, L., and Potier, P., J. Med. Chem., 34, 1991, Rowinsky, E.K. and Donehower, R.C., J. Natl. Cancer Inst., 83, 1991, Guénard, D., Guéritte-Voegelein,F., Dubois, J., and Potier,P., Structure activity relationships of taxol and taxotere analogues, presented at the 2nd Natl. Cancer Institute Workshop on Taxol and Taxus, Alexandria, VA, September E. Tomiak, M.J. Piccart, J. Kerger, D. Devaleriola, E. Tueni, D. Lossignol, S. Lips and M. Bayssas, Eur. J. Cancer, 27 (suppl.2), 1991, F. Gue- ritte-voegelein, D. Guenard, F. Lavelle, M. T. LeGoff, L. Mangatal, P. Potier, J. Med. Chem., 34, 1991, F. Lavelle, C. Fizames, F. Guéritte-Voegelein, D. Guénard and P. Potier, Proc. Am. Ass. Cancer Res., 30, 1989, David G. I. Kingston, Douglas R. Hawkins, and Liza vington, J. Nat. Products, 45, 4, 1982, 466.