INTRODUCTION:

Metoprolol, characterized by its chemical nomenclature as 1-[4-(2-Methoxyethyl) phenoxy]-3-[(propan-2-yl)amino]propan-2-ol, possesses a mol. weight of 267.3 g/mol.¹This compound is acknowledged for its selective blocking action on the β1 receptor.² It is frequently recommended for addressing hypertension, alleviating chest pain arising from insufficient blood flow to the heart, and managing conditions marked by an unusually rapid heart rate.^3,4

This medication is widely utilized in clinical settings to address a range of cardiovascular issues, highlighting its significance in maintaining heart health.¹ Regulatory authorities are meticulously scrutinizing the safety profiles of pharmaceutical compounds. The thorough examination of impurity profiles in pharmaceuticals is increasingly becoming a focal point for ensuring medical safety and the efficacy of active pharmaceutical ingredients (APIs).^5-7 The "International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use" provides specific guidelines regarding impurities present in recently developed pharmaceutical substances, products, and leftover solvents.^8-10 Consequently, there is an increased need for reference standards of impurities, sought after by both regulatory bodies and pharmaceutical firms, as quantifying each impurity in the drug substance is imperative.¹¹The synthesis of Metoprolol impurities reference standards involves key steps such as epoxide ring opening reactions, Fischer esterification reactions, and dimer reactions.¹² Our objective is to produce impurities of API derivatives and characterize them through various methods for impurity identification. Metoprolol impurity EP A, Metoprolol impurity D, Metoprolol impurity F, and N Desisopropyl impurity of Metoprolol are successfully synthesized with favourable yields.

EXPERIMENTAL WORK:

Synthesis of title compound:

In this ongoing investigation, a sequence of compounds of interest was synthesized in the following manner shown in figure 1:

Figure 1: Scheme for synthesis of metoprolol impurity A, D and N-Deisopropoyl.

1) Synthesis of Metoprolol EP impurity A-

Step I

Synthesis of intermediate 2-((4-(2-methoxyethyl) phenoxy)methyl)oxirane.

Inside a clean and dry round-bottom flask (RBF), weigh 0.5 G of the 4-(2-methoxyethyl) phenol. Add 20 mL of dimethylformamide (DMF) to the RBF. Begin stirring the mixture. After 5 to 10 min of stirring, add 0.4 G of potassium carbonate (K₂CO₃) as a base to the reaction mixture. Continue stirring the mixture slowly for 15 min to ensure thorough mixing. Slowly and dropwise, add epichlorohydrin to the reaction mixture. This should be done carefully to prevent any sudden reactions or excessive heat release. Ensure continuous stirring during the addition. Once all the epichlorohydrin has been added, transfer the RBF containing the reaction mixture to an oil bath preheated to 70°C. Attach a reflux condenser to the RBF. Heat the reaction mixture under reflux conditions at a temperature of 70°C for an overnight reaction (approximately 14 h). Ensure that the reaction is maintained at this temperature consistently After 14 H reflux, take out the round-bottom flask (RBF) from oil bath and keep it till it reaches to room temperature. Extract a small sample from the reaction mixture. Analyse the reaction progress by TLC (Thin Layer Chromatography) to check for the presence of desired products.

Step II

Synthesis of 1-Ethylamino-3-(4-(2-methoxyethyl) phenoxy)propan-2-ol.

In a clean and dry round-bottom flask (RBF), weigh 2 G of the 2-((4-(2-methoxyethyl)phenoxy)methyl)oxirane (Synthesis as per Step I). Add 20 mL of methanol to the RBF. Begin stirring the mixture. After 5 to 10 min of stirring, add 0.7 ml of ethylamine. Continue stirring the mixture slowly for 15 minutes to ensure thorough mixing. This should be done carefully to prevent any sudden reactions or excessive heat release. Ensure continuous stirring during the addition. Once all the ethylamine has been added, transfer the RBF containing the reaction mixture to an oil bath preheated to 80°C. Attach a reflux condenser to the RBF. Reflux the reaction mixture at 80°C for an approximately 14h. Ensure that the reaction is maintained at this temperature consistently After 14 h of reflux, Take the round-bottom flask (RBF) out of oil bath and keep it till it reaches to room temperature. Extract a small portion of the reaction mixture. Analyse the reaction progress by TLC (Thin Layer Chromatography) to check for the presence of desired products.

2) Synthesis of Metoprolol Impurity D-

Step I:

Same as per Impurity A Step I.

Step II:

Synthesis of 3-(4-(2-methoxyethyl)phenoxy)propane-1,2-diol.

In a round-bottom flask that is both clean and dry, weigh 1 G of the 2-((4-(2-methoxyethyl) phenoxy) methyl) oxirane (Synthesis as per Impurity A Step I). Add 20 mL of dimethylformamide (DMF) to the RBF. Begin stirring the mixture. After 5 to 10 min of stirring, add 0.5 grams of Aq. NaOH as a base to the reaction mixture. Continue stirring the mixture slowly for 15 min to ensure thorough mixing. Attach a reflux condenser to the RBF. Heat the reaction mixture under reflux conditions at 70°C for an 12 h. Ensure that the reaction is maintained at this temperature consistently After 14 H of reflux, Take out the round-bottom flask (RBF) from oil and keep it till it reaches to room temperature. Obtain a small sample from the reaction mixture. Analyse the reaction progress by TLC (Thin Layer Chromatography) to check for the presence of desired products.

3) Synthesis of N Desisopropyl Metoprolol:

Step I:

Same as per Impurity A Step I

Step II:

Synthesis of 1-amino-3-(4-(2-methoxyethyl)phenoxy) propan-2-ol

Take a clean and dry Round-Bottom Flask (RBF) Add 2 G the intermediate 2-((4-(2-methoxyethyl)phenoxy) methyl)oxirane(Synthesis as per Impurity A Step I)into the RBF. Add 20mL of the solvent to the RBF containing the product. Ensure that the solvent is measured accurately. Carefully add Ammonia to the RBF. The amount of Ammonia should be appropriate for your specific reaction conditions. Ensure that safety measures are in place, such as proper ventilation and personal protective equipment, when handling Ammonia. Stir the mixture thoroughly using a magnetic stirrer or a suitable stirring mechanism to ensure proper mixing of the components. Set up a gas purging apparatus for the additional addition of ammonia gas. Ensure that the apparatus is securely connected to the RBF. Allow the reaction to proceed overnight. It is important to maintain a controlled and stable environment during this time to ensure the success of the reaction. After 12 H of reaction time, take a Thin-Layer Chromatography (TLC) sample to monitor the progress of the reaction and to check for the completion of the reaction. Analyse the TLC results to determine the extent of the reaction and whether further steps are needed.

4) Synthesis of Metoprolol F-

Synthesis of intermediate 2- (phenoxymethyl) oxirane

In a clean and dry round-bottom flask (RBF), weigh 5 G of the phenol. Add 50mL of dimethylformamide (DMF) to the RBF. Begin stirring the mixture at cooling condition. After 5 to 10 minutes of stirring, add 7.4 G of potassium carbonate (K₂CO₃) as a base to the reaction mixture. Continue stirring the mixture slowly for 15 min to ensure thorough mixing. Slowly and dropwise, add epichlorohydrin to the reaction mixture. This should be done carefully to prevent any sudden reactions or excessive heat release. Ensure continuous stirring during the addition. Once all the epichlorohydrin has been added, transfer the RBF containing the reaction mixture to an oil bath preheated to 80°C. Attach a reflux condenser to the RBF. Heat the reaction mixture under reflux conditions at 80°C for approximately 12 hour. Take the round-bottom flask (RBF) out of oil bath and keep it till it reaches to room temperature. Extract a small portion of the reaction mixture. Analyse the reaction progress by TLC (Thin Layer Chromatography) to check for the presence of desired products.

Synthesis of 1-(isopropylamino)-3-phenoxypropan-2-ol: In a round-bottom flask (RBF) that is both clean and dry weigh 4.92 G of the 2- (phenoxymethyl) oxirane. Add 50 mL of dimethylformamide (DMF) to the RBF. Begin stirring the mixture at cooling condition. After 5 to 10 min of stirring, add 7.4 G of potassium carbonate (K₂CO₃) as a base to the reaction mixture. Continue stirring the mixture slowly for 15 min to ensure thorough mixing. Slowly and dropwise, add epichlorohydrin to the reaction mixture. This should be done carefully to prevent any sudden reactions or excessive heat release. Ensure continuous stirring during the addition. Once all the epichlorohydrin has been added, transfer the RBF containing the reaction mixture to an oil bath preheated to 80°C. Attach a reflux condenser to the RBF. Reflux the reaction mixture at 80°C for approximately 12 hour. Take the round-bottom flask (RBF) out of oil bath and keep it till it reaches to room temperature. Extract a small sample of the reaction mixture. Analyse the reaction progress by TLC (Thin Layer Chromatography) to check for the presence of desired products

Characterization of Designed Compound:

The Spectral data, including Infrared (IR)^13-14, Nuclear-Magnetic-Resonance (NMR)¹⁵, and Mass Spectrometry (MASS)¹⁶, were employed to verify the structures of the synthesized compounds, while microanalysis (e.g HPLC-UV, SEM etc)^17-19confirmed their purity in vitro as well as in vivo.²⁰The melting points of the synthesized compounds were measured using capillaries on Labronics LT-115 digital melting point and boiling point apparatus and are uncorrected. The TLC analyses were performed on an aluminium sheet pre-coated with silica gel. Visualization of spots on TLC plates was accomplished with UV light.^21-23The JASCO-FTIR 4100 spectrophotometers were utilized to record the IR spectra of the synthesized compounds, employing KBr (with υmax values in cm-¹). The NMR spectra of the synthesized compounds were obtained in CDCl₃ (unless otherwise stated) and DMSO, utilizing TMS as the internal reference (Chemical shift in δ, ppm). The measurements were conducted using the MERCURY VARIAN 500 MHz instrument and the MERCURY VARIAN 400 MHz instrument. The Mass spectra of the compounds were acquired using the Bruker Impact HD instrument.

Purification through chemical separation relies significantly on the foundational technique of column chromatography. This method involves introducing a liquid mobile phase into a column containing a solid stationary phase. As the compound mixture descends into the column, compounds undergo separation from one another, driven by distinct interactions with the stationary phase. The effectiveness of column chromatography lies in its essential role for the isolation and purification of chemicals, making it a common place tool in both research and industrial applications.

RESULTS AND DISCUSSION:

Characterization of designed compound-

Spectral data of Metoprolol Impurity A:

1H-NMR -7.11-7.13(d,1H, CH), 6.83-6.86(s,2H, CH), 3.34 (s,3H, CH2), 3.53-3.57(t,2H, CH2), 3.95-3.96(t,2H, CH2), 2.70-2.74(m,2H, CH), 2.80-2.83(t,2H, CH2), 2.85-2.89(m,1H, CH), 4.06-4.08(broad,1H, OH), 1.12-1.16(m,3H, CH3), 2.41(broad,3H, CH2)

FTIR Spectra- 3553.08 (1) (O-H), 3282.25(2) (N-H), 1174.44 (26) (C-N), 1194.69 (25) (C-O)

Spectral data of Metoprolol Impurity D:

¹H-NMR -7.13-7.17(d,2H, CH), 6.81-6.85(d,2H, CH), 4.98-4.01(m,1H, CH), 3.54-3.57(t,2H, CH₂), 3.34(s,3H, CH₃), 2.80-2.84(t, 2H, CH₂), 4.10-4.13(dd,2H, CH₂), 4.5-4.62(dd,2H, CH₂)

FTIR spectra –3481.85 (1) (O-H), 3392.55 (2) (O-H), 1609.31 (17) (C=C), 1115.62 (30) (C-O)

Spectral data of Metoprolol N Desisopropyl Impurity:

¹H-NMR -7.10-7.12 (d,2H, CH₂), 6.82-6.84 (d,2H, CH₂), 3.45-3.48(t,2H, CH₂), 3.22 (s,3H, CH₂), 2.70-2.73 (t,2H, CH₂), 3.66-3.79 (m, 1H, CH), 3.78-3.90 (m,2H, CH₂), NH₂ – Proton merge in DMSO

FTIR spectra –3647.7 (8) (O-H), 3222.47(9) (N-H), 1637.27(16) (C=C), 1177.33(25) (C-O)

Spectral data of Metoprolol Impurity F:

¹H-NMR -7.27-7.31(d,2H, CH), 6.95-6.97(d, 1H, CH), 6.90-6.93(d,2H,3H), 3.94-4.0(t, 2H, CH₂), 4.05-4.08(m, 1H, CH), 2.86-2.94(M, 2H, CH₂), 2.66-2.79(M, 1H, CH), 2.59(broad, 1H, OH, NH)

FTIR spectra –3647.7 (1) (O-H), 3315.03(5) (N-H), 1624.98(16) (C=C), 1147.47(27) (C-O)

SUMMARY AND CONCLUSION:

Impurities persist throughout the synthesis of drugs and active pharmaceutical ingredients (APIs), even in trace amounts. Despite their minimal presence, chemically produced impurities hold significant market value within the pharmaceutical industry. In the synthesis process of Metoprolol, these impurities play a crucial role as reference standards. Therefore, our research study focused on synthesizing four Metoprolol impurities, namely Metoprolol Impurity-A, Metoprolol Impurity-D, Metoprolol Impurity-F and N-Desisopropyl impurity of Metoprolol.

Metoprolol serves various roles in the pharmaceutical industry, particularly in treating heart diseases, contributing significantly to its market value. The purification of impurities was conducted using column chromatography techniques. A dual-method approach was employed to confirm the identity of these compounds, involving single-spot TLC and comprehensive spectral studies utilizing techniques such as IR, ¹H NMR, and mass spectroscopy.We synthesized impurities related to emergency cardiovascular drugs, enhancing their market value in the pharmaceutical industry due to the critical role they play in ensuring the safety of potent drugs.

ACKNOWLEDGMENTS:

We would like to extend our appreciation to Anasyn Lab Pvt. Ltd. for providing essential synthetic requirements and analytical information regarding the compound. Their invaluable support and cooperation were instrumental throughout the implementation of this project.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 05.04.2024 Revised on 16.08.2024

Accepted on 20.10.2024 Published on 27.03.2025

Available online from March 27, 2025

Research J. Pharmacy and Technology. 2025;18(3):1128-1133.

DOI: 10.52711/0974-360X.2025.00162

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Sr. No.	Common Name	IUPAC Name	Appearance	Yield (%)
1	Impurity-A	1-(ethylamino)-3-[4-(2-methoxyethyl)phenoxy]propan-2-ol.	White Solid	60.97%
2	Impurity-D	3-[4-(2-methoxyethyl)phenoxy]propane-1,2-diol.	Light Green Solid	68.88%
3	N -deisopropyl Impurity	1-amino-3-[4-(2-methoxyethyl)phenoxy]propan-2-ol.	White Solid	48%
4	Impurity F	1-phenoxy-3-[(propan-2-yl)amino]propan-2-ol	White Solid	79.52%