Ind. Eng. Chem. Res. 2008, 47, 9201–9205
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APPLIED CHEMISTRY Reaction of Finasteride Intermediate with Benzeneseleninic Anhydride: An In-Depth Study Ch. Suchitra,† Kalyani Maitra,† Dilip Raut,† Lakshmi Shilpa,† Hanmandlu Dodla,‡ Yalavarti Ravindrakumar, Apurba Bhattacharya,† Mulukutla V. Suryanarayana,† and Gautam Samanta*,† Dr. Reddy’s Laboratories Limited, IPDO InnoVation Plaza, Bachupally, Hyderabad, India, and Dr. Reddy’s Laboratories Limited, CTO-2, Bollaram, Hyderabad, India
One of the intermediates of finasteride was oxidized by the reaction of benzeneseleninic (BS) anhydride in refluxing toluene for 16 h. Sometimes, the desired product was obtained, and sometimes, there was no yield or very low yield at the end of the reaction time. To obtain a better understanding of such variations, laboratory syntheses were conducted with commercial BS anhydride, pure BS anhydride, pure BS acid, and mixtures of anhydride and acid samples. During the reaction, the intermediate reaction masses were analyzed by highperformance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) at predetermined times and correlated with differential scanning calorimetry (DSC) and X-ray powder diffraction (XRPD) samples. The DSC and XRPD data indicated that commercial BS anhydride samples did not always consist of the pure anhydride form. When the BS acid was present in the anhydride sample, the reaction rate was lower than that of the pure BS anhydride sample. Introduction Finasteride is an antiandrogen that acts by inhibiting type II 5R-reductase,1 the enzyme that converts testosterone to dihydrotestosterone (DHT). It is used as a treatment in benign prostatic hyperplasia (BPH) in low doses and prostate cancer in high doses.2 Finasteride was approved initially in 1992 as Proscar, a treatment for prostate enlargement, but the sponsor of the drug had studied 1 mg of finasteride and demonstrated hair growth in male pattern hair loss.3 On December 22, 1997, the FDA approved finasteride to treat male pattern hair loss. Benzeneseleninic (BS) anhydride has been used as an oxidant for different organic compounds.4,5 It has also been reported that BS anhydride is an excellent reagent for the dehydrogenation of ketones.6-8 The yields of individual reactions depend on the reaction conditions and substrates. Dragojlovic8 reported that an acceptable yield was obtained only within a relatively narrow range of conditions and that excess reagent, increased temperature, or extended reaction times lowered the yield. In the preparation of finasteride [17-β-(n-tertiary-butyl carbamoyl)-3-oxo-4-aza-5-R-androstane] (FIN-1) was reacted with BS anhydride in a mole ratio of 1:2.6 in refluxing toluene for 16 h (Figure 1). The reaction time was optimized based on the amount of starting material (FIN-1) present in the reaction mixture. It was observed that FIN was formed before 16 h, but it contained fair amount of FIN-1. After 16 h of refluxing, the amount of FIN-1 was reduced to below the detection limit. Recently, the yield of the FIN varied from batch to batch using the same FIN-1 starting material. Sometimes, FIN was obtained * To whom correspondence should be addressed. Address: Dr. Reddy’s Laboratories Limited, Survey No. 42, 45, 46, & 54; Bachupally, R. R. District, Hyderabad, A. P., India 500 072. E-mail: gautams@ drreddys.com and
[email protected]. Tel.: 91 40 4434 600ext 6437. Fax: 91 40 44346164. † IPDO Innovation Plaza. ‡ CTO-2.
in the desired quantity, whereas there were some cases where the yield was poor or no product was formed. It was postulated that BS anhydride could be responsible for such inconsistencies. To investigate the inconsistent behavior, several experiments were carried out using differential scanning calorimetry (DSC) and X-ray powder diffraction (XRPD) with pure BS anhydride, pure BS acid, and commercially available BS anhydride samples purchased from local vendors. Laboratory syntheses were carried out using different BS anhydride samples, and the reaction course was monitored by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) at predetermined times. The main objective of this study was to interrogate the causes of the low or nonexistent yields of FIN using the specific batches of BS anhydride samples. To meet the main objective, the following studies were carried out: (1) Commercial BS anhydride samples were analyzed by DSC and XRPD prior to any laboratory analysis. Pure BS anhydride and BS acid samples were also analyzed. (2) Laboratory syntheses were conducted using pure BS anhydride, pure BS acid, different ratios of anhydride and acid mixtures, and commercial BS anhydride samples. (3) HPLC and LC-MS methods were used for quantification and characterization of the intermediate products. (4) The inference was drawn by interpreting the DSC, XRPD, HPLC, and LC-MS data. Experimental Section Materials. Commercial BS anhydride was purchased from two vendors, Omkar Chemicals, Maharashtra, India, and SA Pharmachem, Mumbai, India. For laboratory analysis, samples were packed in a nitrogen-sealed plastic bag. FIN-1 was the in-house product of Dr. Reddy’s Laboratory, Hyderabad, India. Samples of pure BS anhydride (>98% pure) and BS acid (99% pure) were purchased from Fluka, Deisenhofen, Germany.
10.1021/ie800530c CCC: $40.75 2008 American Chemical Society Published on Web 11/11/2008
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Figure 1. Synthetic scheme for the conversion of FIN-1 to FIN.
Laboratory Synthesis. Typically, in a 1-L three-neck reaction vessel containing 276 mL of toluene under nitrogen, 50 g of BS anhydride was charged and then azeotropically refluxed with stirring for 1 h. Next, 20 g of FIN-1 was added at 65 ( 5 °C under nitrogen atmosphere, and the mixture was azeotropically refluxed for an additional 16 h. Before charging of the reagents into the reactor, the moisture contents of toluene and starting material (FIN-1) were measured and were found to be 0.03-0.08% and 0.1-0.15%, respectively. After completion of the reaction, the product was washed with water, and the organic layer (toluene) was distilled off under vacuum. The residue was washed with cyclohexane several times to obtain pure FIN. The reaction course was monitored by collecting sample mixtures at different time intervals using two commercial BS anhydride samples (A and B). To better understand the chemical kinetics, four additional experiments were performed using pure BS anhydride, pure BS acid, and two mixtures of anhydride and acid samples (25:75 and 75:25). The BS acid/anhydride/mixture sample (1.25 g) was refluxed azeotropically in a 100-mL round-bottom flask with 15 mL of toluene for 1 h and then cooled to 50-60 °C. At this temperature, 0.5 g of FIN-1 was charged and continued to reflux. One milliliter of the reaction mixture was taken into a small vial at predetermined time intervals. After collection of the sample, 1 mL of hot water (90-95 °C) was added to the vial and shaken well. The water layer was removed, and the whole process was repeated twice. Finally, the toluene was distilled off using N2 flow and then analyzed by HPLC. DSC Analysis. All BS anhydride samples, including pure BS anhydride, were analyzed by DSC. The analyses were performed on a DSC Pyris 6 instrument (Perkin-Elmer, Wellesley, MA) under a nitrogen gas purge at a flow of 50 mL/min. The sample was heated from 40 to 200 °C at 10 °C/min. XRPD Analysis. X-ray powder diffraction studies were performed by exposing the sample to Cu KR radiation (45 kV × 35 mA) in a Bruker AXS D8 Advance wide-angle X-ray diffractometer (Bruker AXS GmbH, Karlsruhe, Germany). The instrument was operated in the continuous scan mode in increments of 0.008° 2θ. The angular range was 3-45° 2θ, and counts were accumulated for 0.0165 s at each step. Data acquisition and analysis were performed with Bruker DIFFRAC plus EVA software. HPLC Analysis of Intermediate Samples. The HPLC system consisted of Shimadzu SCL-10AVP with LC-10AT VP delivery pumps, an SPD-10A VP UV-vis detector, an SIL10AD VP auto injector, a DGU-12A degasser, a CTO-10AS VP column oven, and Class-VP series software. The mobile phase was prepared using 0.25% orthophosphoric acid (sample A) and acetonitrile (water/acetonitrile ) 10:90) (sample B). The gradient method was used. The flow rate was 0.8 mL/min. Separation was achieved using a Novapak C18 (250 × 4.6 mm × 4 µm) column. A detailed description of the HPLC analysis is available in the literature.9
LC-MS Analysis of Intermediate Samples. The LC-MS instrument used for analysis was an Applied Biosystems, 4000 Q-trap LC/MS/MS apparatus with an Agilent 1100 series chromatograph, a G1379A degasser, a G1312A binary pump, and a G1316A column compartment. The instrument was equipped with an electrospray ionization (ESI) source. The same column as above was used. Inert PEEK tubing was used for all mobile-phase connections. A mobile phase composed of 0.01% trifluoroacetic acid (TFA) and acetonitrile (90%) with a flow rate 0.8 mL/min was used. Results and Discussion Laboratory Synthesis and Analysis. The first laboratory synthesis was performed using commercial BS anhydride sample A. The intermediate products were collected at 4-, 8-, 12-, and 16-h intervals for analysis. All samples were analyzed by HPLC and LC-MS. The BS anhydride sample that was collected before charging was sealed in a polyethylene packet under nitrogen and analyzed immediately in the laboratory. The chromatogram of the 4-h sample using BS anhydride sample A is shown in Figure 2, which indicates that the samples contained many components including FIN, BS acid, and diphenyl diselenides (DPDS). The main peaks were characterized by analyzing pure samples. FIN-1 and toluene eluted at the same retention time (RT ≈ 10.6 min). For confirmation, LC-MS was performed, and the LC-MS data confirmed that the small peak was due to the presence of toluene in the sample. A typical chromatogram (LC-MS analysis) of the 4-h sample is shown in Figure 3. The total ion chromatogram (TIC) of the sample and typical mass spectra for BSA, FIN, and DPDS are provided in the Supporting Information (SI 1). The LC-MS data confirmed the presence of major components in the intermediate reaction masses. It is important to note that FIN-1 was not detected in the 4-h sample. This means that reaction was completed within 4 h. The yield of FIN at 4 h was ∼20%. The product profiles at different reaction times are reported in Table 1, which indicates that the area percentage of FIN was reduced from 10.95% to 6.84% as the reaction time increased to 4 h. The yield was also reduced from 20% to 11% at 16 h. The concentrations of BSA decreased with increasing reaction time. On the other hand, the concentrations of DPDS increased from 48% to 81% as the reaction time increased from 4 to 16 h. From this experiment, it is difficult to draw any conclusion on the reaction kinetics because the conversion of FIN-1 to FIN was completed within the first few hours. Therefore, another experiment was conducted under the same experimental conditions using BS anhydride sample B, and samples were collected for analysis at 1, 2, 3, 4, 5, 6, and 16 h. The results are also summarized in Table 1, which indicates that, after 1 h of reaction, FIN was formed (18.88% yield), but it contained fair amount of FIN-1 (9.46% area). As the reaction
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Figure 2. HPLC chromatogram for 4-h sample produced by the reaction of BS anhydride (sample A) using orthophosphoric acid method.
Figure 3. Chromatogram of the 4-h sample produced by the reaction of BS anhydride (sample A) at λ ) 210 nm using LC-MS analysis.
time increased, the amount of FIN-1 decreased (FIN-1 was confirmed by LC-MS analysis), and at 16 h, FIN-1 was not detected. On the other hand, the amount of FIN increased within 1 h (18.88% yield) to 5 h (52.35% yield) and then decreased with time. It is important to note that, in this case, a significant amount of FIN-1 was detected in the 4- and 6-h samples, whereas in the first experiment, FIN-1 was not detected in the 4-h sample. Thus, the rate of reaction in the second experiment using BS anhydride sample B was lower than that in the first experiment with BS anhydride sample A. Although the reaction took a long time with BS anhydride sample B, the yield was
higher, presumably because the product had less time to decompose after its formation. To understand the low reaction rate, DSC and XRPD analyses were performed for both BS anhydride samples (A and B) and compared. The DSC analyses of the BS anhydride samples A and B are shown in Figures 4 and 5, respectively, which indicate that the two BS anhydride samples were not identical. A sharp endothermic peak (Figure 4) for BS anhydride sample A was observed at 174.8 °C. The endothermic peak is due to melting of the compound. There was no characteristic peak for BS acid within the range 121-124 °C. The DSC pattern for BS
9204 Ind. Eng. Chem. Res., Vol. 47, No. 23, 2008 Table 1. Area Percentages for BSA, FIN-1, FIN, and DPDS in Intermediate Samples at Different Times of Reaction Using Two BS Anhydride Samples (A and B) Measured by HPLC experiment 1 (with BS anhydride A)
experiment 2 (with BS anhydride B)
sampling time (h)
BSA
FIN
DPDS
BSA
FIN
FIN-1
DPDS
1 2 3 4 5 6 8 12 16
NAa NA NA 29.56 NA NA 13.06 4.69 0.22
NA NA NA 10.95 NA NA 7.67 6.51 6.48
NA NA NA 48.71 NA NA 81.13 81.52 84.20
74.87 69 60.89 55.36 49.58 48.81 NA NA 3.57
5.67 9.09 12.48 15 15.72 15.11 NA NA 11.98
9.46 6.88 4.76 3.67 2.34 1.62 NA NA NDb
9.2 13.19 19.02 23.05 28.28 29.7 NA NA 77.4
a
NA ) not available. b ND ) not detected.
Figure 6. XRPD patterns for BS anhydride samples A and B.
Figure 4. DSC scan for BS anhydride sample A. Figure 7. XRPD pattern for pure BS anhydride and pure BS acid samples.
Figure 5. DSC scan for BS anhydride sample B.
anhydride sample B (Figure 5) showed a prominent endothermic peak for BS acid at 121.1 °C and endothermic peak for BS anhydride (174.5 °C). Therefore, BS anhydride sample B might be a pure BS acid or a mixture of BS anhydride and BS acid, whereas sample A was pure anhydride. The DSC pattern for pure BS anhydride was comparable to that of commercial BS anhydride sample A. The DSC pattern for the pure BS anhydride sample is shown in the Supporting Information (SI 2). The slow reaction kinetics of the second experiment might be due to the poor quality of BS anhydride sample B. XRPD analyses were conducted for all BS anhydride samples. The X-ray diffraction patterns of two BS anhydride samples (A and B) prepared by physical grinding at room temperature under nitrogen atmosphere are shown in Figure 6. The overlaid diffractograms indicate significant differences between the two samples (A and B). For BS anhydride sample A, there were distinct peaks at 7.454°, 7.861°, and 10.821°, whereas no such characteristic peaks were found for BS anhydride sample B. These experiments clearly indicate that these two BS anhydride samples were not same in nature. To characterize the two
different BS anhydride samples, XRPD analyses were performed using pure BS anhydride and pure BS acid samples. The overlaid XRPD patterns of both pure samples are shown in Figure 7, which indicates that pure anhydride was identical to BS anhydride sample A and pure acid was superimposed on BS anhydride sample B. From this experiment, it was concluded that BS anhydride sample B was present in acid form or a mixture of acid and anhydride forms. The quantification method for the BS acid in BS anhydride samples will be discussed in a forthcoming publication. The slow reaction kinetic might be due to the presence of BS acid in BS anhydride sample B. For a better understanding of the chemical kinetics, four more experiments were performed with two physical mixtures of pure BS acid and pure BS anhydride samples (25:75 and 75:25), including pure BS anhydride and pure BS acid samples. The reaction masses at predetermined times were analyzed by HPLC. The ratios (areas) of FIN-1 and FIN for each experiment are plotted against time (in hours) in Figure 8. If more FIN is present in the mixture, the value should be small. That means that more FIN-1 has been converted to FIN within the time of reaction. After 1 h of reaction, the results showed that the value of FIN-1/FIN in the reaction of 100% BS acid was much higher than the values with the reaction of 100% BS anhydride and the mixtures of BS acid and BS anhydride. A decreasing trend of FIN-1/FIN ratios was obtained over the course of the reaction. The trend was as follows: 100% BS acid > 75% BS acid + 25% BS anhydride > 25% BS acid + 75% BS anhydride > 100% BS anhydride. On the other hand, it could be said that, with increasing BS anhydride or decreasing BS acid, more FIN was formed within a short period of time. These results clearly indicate that BS anhydride is more reactive than free BS acid,
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Supporting Information Available: Total ion chromatogram (TIC) of sample A and typical mass spectra for BSA, FIN, and DPDS. This material is available free of charge via the Internet at http://pubs.acs.org. Literature Cited
Figure 8. FIN-1/FIN ratios for each experiment with pure BS acid, pure anhydride, 25:75 acid/anhydride mixture, and 75:25 acid/anhydride mixture at different times.
and the presence of BS acid reduces the reaction rate. Therefore, before the synthesis of finasteride, the quality of the BS anhydride reagent should be monitored. Conclusion Based on the experimental results, it was concluded that pure BS anhydride is more reactive than free BS acid. An increase in the amount of of free acid in an anhydride sample decreases the reaction rate. Prolonged heating also reduces the yield of FIN. The final conclusion from these experiments is that the reaction should be conducted with pure BS anhydride as a costeffective process. Acknowledgment The authors gratefully acknowledge Dr. Reddy’s Laboratories, Hyderabad, India (Communication No. IPDOIPM-00132) to provide all supports for this work. The authors are indebted to AR&D (IPDO), CTO-2, and CEO-Polymer division for their cooperation.
(1) Geller, J. Effect of Finasteride, a 5R-Reductase Inhibitor on Prostate Tissue Androgens and Prostate-Specific Antigen. J. Clin. Endocrinol. Metab 1990, 71, 1552. (2) Nickel, J. C.; Andersen, J. T. Update on the Use of Finasteride in Benign Prostatic Hyperplasia: Long-Term Results. In Recent AdVances in Prostate Cancer and BPH; Schroder, F. H., Ed.; Parthenon Publishing Group Inc.: New York, 1997. (3) Shapiro, J.; Kaufman, K. D. Use of Finasteride in the Treatment of Men with Androgenetic Alopecia (Male Pattern Hair Loss). J. InVest. Dermatol. Symp. Proc. 2003, 8, 20. (4) Barton, D. H. R.; choi, S.-Y.; Liu, W.; Smith, J. A. Oxidation of Phenylhydrozones with Benzeneseleninic Anhydride: A New Mechanistically Interesting Observation. Mol. Online 1998, 2, 22. (5) Clayton, M. D.; Marcinow, Z.; Rabideau, P. W. Benzeneseleninic Anhydride Oxidation of 1,2-Diarylethanes and 1,2-Diarylethylenes to 1,2Diaryldiketones. Tetrahedron Lett. 1998, 39, 9127. (6) Barton, D. H. R.; Lester, D. J.; Ley, S. V. Dehydrogenation of Steroidal and Triterpenoid Ketones Using Benzeneseleninic Anhydride. J. Chem. Soc., Perkin Trans. 1 1980, 2209. (7) Morzycki, J. W.; Obidzin´ska, J. On the Reaction of A-nor-5acholestan-2-one with Benzeneseleninic Anhydride. Can. J. Chem. 1991, 69, 790. (8) Dragojlovic, V. Preparation of Cyclopentenones by Benzeneseleninic Anhydride Oxidation of Cyclopentanones. J. Chem. Res. 1999, 256. (9) Cendrowska, I.; Buszewski, B. Determination of Finasteride and Related Compounds by Reversed-Phase High Performance Liquid Chromatography. I. Choosing the Mobile Phase Composition. J. Liq. Chromatogr. Relat. Technol. 1999, 22, 2259.
ReceiVed for reView April 3, 2008 ReVised manuscript receiVed September 1, 2008 Accepted September 4, 2008 IE800530C
