Identification and Quantitative Analysis of 2-Phenylethanol as Major Impurity in Bromadol by HPLC

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Initial studies showed that Bromadol was around 10’000 times the strength of morphine ^[2] in animal models. Recent research ^[3] assigned a value of x504 morphine for the more active trans Bromadol isomer. The antinociceptive ED₅₀in mouse hot plate of Bromadol is 13.4 μg/kg ^[3] and the binding K_i values for μ-opioidreceptor of the Bromadol is 1.49 nM ^[3]. Recently, Bromadol was prepared and used as regulators for the nociceptin/orphanin FQ ligand ORL-1 receptor system ^[4].

Bromadol is also the lead compound of 4-amino-4-arylcyclohexanone derivatives ^{[5, 6, 7, 8, 9]} which can serve as a potent class of analgesics ^{[3, 6, 7]}. The structure-activity relationships (SAR's) of this series compounds has been discussed ^{[10, 11]}. p-halo substitutions optimize the ability of the compounds to gain access to the analgesic receptors, whereas m-OH substitution induced narcotic antagonist activity ^[10]. Trans conformation and hydrophobicity of the substituents at the C atom possessing the OH group in the cyclohexanol nucleus were most important for the analgesic activity ^[11].

Until now, many literatures ^{[2, 4, 7, 12, 13, 14]} have been reported concerning about the synthesis of Bromadol. The most widely used synthesis route is outlined in scheme 1.

Scheme 1. Most widely used synthesis route of Bromadol

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As Bromadol could serve as a powerful and versatile agent in medicinal chemistry, it is of great importance to identify the impurities in Bromadol and establish a quality control methodology for production. However, few literatures have been reported concerning about this issue until now.

In this paper, we try to employ HPLC to identify and quantitative analysis of impurities in Bromadol (The Bromadol sample was donated by Beijing Realchem Technology Co., Ltd as a gift).

2. Experimental

Firstly, the commercial Bromadol sample was dissolved in absolute methanol and the concentration was shown in Table 1.

Table 1. commercial Bromadol sample solution

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The solution was then subjected to HPLC analysis. The HPLC spectrum and the analysis result were shown in Figure 2 and Table 2 respectively.

Figure 2. HPLC spectrum of the commercial Bromadol sample solution

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Table 2. HPLC analysis data of the commercial Bromadol sample solution

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As shown in Figure 2 and Table 2, the retention time of Bromadol was 17.392 min with peak area percentage 95.692% (No.7, Table 2). Many impurity peaks can also be observed. The retention times of the main impurity peaks were 4.379 min, 7.312 min, 11.395 min, 14.972 min respectively.

Subsequently, we tried to identify the main impurities in the commercial Bromadol sample. As impurities may always come from the last synthesis step, the most widely used synthesis route of Bromadol (Scheme 1.) was carefully studied. We speculate that the main impurity may be 2-phenylethanol which may result from hydrolysis of the phenethylmagnesium bromide.

Table 3. 2-phenylethanol standard solution

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A standard solution of 2-phenylethanol in absolute methanol was prepared and the concentration was shown in Table 3.

The standard 2-phenylethanol solution was subjected to HPLC under the same analysis conditions which have been employed for the commercial Bromadol sample solution. The HPLC spectrum and analysis result of the standard 2-phenylethanol solution were shown in Figure 3 and Table 4 respectively.

Figure 3. HPLC spectrum of 2-phenylethanol standard solution

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Table 4. HPLC analysis data of 2-phenylethanol standard solution

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As shown in Figure 3 and Table 4, the retention time of 2-phenylethanol was 4.375 min with peak area percentage 96.156% (No. 2, Table 4). The retention time of 2-phenylethanol was same as one of the main impurities in the commercial Bromadol sample (see Figure 2). This result indicated that one of the main impurities in the commercial Bromadol sample was 2-phenylethanol.

Next, we tried to analyze the 2-phenylethanol impurity in the commercial Bromadol sample. The commercial Bromadol sample solution and the standard 2-phenylethanol solution were subjected to HPLC analysis for 3 times under the same conditions which have been employed previously. The peak area of 2-phenylethanol of the two solutions were shown in Table 5.

Table 5. Peak area of 2-phenylethanol in two solutions

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With the peak area data of 2-phenylethanol in Table 1, Table 3 and Table 5, the concentration of impurity 2-phenylethanol in commercial Bromadol sample solution was calculated to be 0.0987 mg/ml and the 2-phenylethanol accounts for 9.6% of the commercial Bromadol sample by weight.

As 2-phenylethanol is a colorless oil with boiling point of 219°C, we speculate that it could be removed from Bromadol by drying under vacuum without leading to decomposition of Bromadol.

0.5 gram of the commercial Bromadol sample was placed into vacuum drying oven at 45°C for 12 h. After drying under vacuum, the Bromadol sample was dissolved in absolute methanol and the concentration was shown in Table 6.

Table 6. Dried Bromadol sample solution

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The dried Bromadol sample solution was subjected to HPLC under the same analysis conditions employed previously. The HPLC spectrum and the analysis data were shown in Figure 4 and Table 7 respectively.

Figure 4. HPLC spectrum of dried Bromadol sample solution

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Table 7. HPLC analysis data of dried Bromadol sample solution

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Figure 5. H-NMR spectrum of the dried Bromadol sample

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